Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. 171 65

The platelet glycoprotein (GP) IIb-IIIa complex figures prominently as an immunogen in autoimmune (idiopathic) thrombocytopenic purpura (ITP). 2E7 is a human monoclonal IgM autoantibody, derived from splenocytes of a patient with ITP, that recognizes a specific octapeptide amino acid sequence, Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser, on the heavy chain of GPIIb. This represents the first precise identification of an epitope on GPIIb-IIIa recognized by a human antibody. In this study, we have isolated total mRNA from 2E7, synthesized the corresponding cDNA using reverse transcriptase, and amplified the immunoglobulin mu and kappa chain cDNA by the Taq 1 polymerase chain reaction (PCR) using specific primers. The 2E7 mu chain variable region is encoded by a VH3 gene segment that is 98% homologous to the germline gene VH1.9III, a D-gene that is not homologous to any of the germline D-genes reported to date, and a JH6 gene segment that is essentially germline. The heavy-chain sequence, save for the unique D-gene, is similar to that of a number of human autoantibodies. The 2E7 kappa variable region is encoded by a Vk1 gene segment linked to a Jk1 gene segment. The Vk1 sequence of 2E7, with the exception of one nucleotide, is identical to that of autoantibody HF2-1/17, a prototype of SLE-associated anti-DNA autoantibodies bearing the 16/6 idiotype. The single base substitution results in a relatively conservative exchange of Asp for Glu at position 70 of the protein sequence. Despite this near identity in sequence, 2E7 does not bind to either single-stranded or double-stranded DNA. From these results, we conclude that specificity of 2E7 is likely to reside in either or both the D-JH region (CDR3) of the mu chain and the Jk region (CDR3) of the kappa chain. In addition to the identification of a novel D-gene, we also provide evidence that the 2E7 VHIII gene is probably a prototype of a VHIII subfamily, that the germline Vk1 gene shared by 2E7 and autoantibodies of the 16/6 idiotype probably represents a separate Vk family, and that this Vk gene cannot itself attribute specificity for DNA.
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PMID:Nucleotide sequence of the human autoantibody 2E7 specific for the platelet integrin IIb heavy chain. 171 98

RNA sequences for the V regions of human hybridoma-produced autoantibodies were determined by primer extension with reverse transcriptase. The sequencing of IgM autoantibodies from a leprosy patient revealed examples of recurrent use of V region gene segments in different autoantibodies from this patient and a previously studied patient with SLE. Moreover, several gene segments used in these autoantibodies show little alteration from germ-line sequences. mAb TH3, from a patient with leprosy, binds denatured DNA and poly(dT). The center of its H chain CDR35 has a sequence identical to that found previously in two anti-DNA antibodies from a lupus patient; these identities and their overlapping with two other published sequences define a human D-gene segment of approximately 25 nucleotides. Autoantibody TH9, from a leprosy patient, does not bind DNA. Its VH sequence has 87% identity with a VHI anti-DNA antibody, but differs from it markedly in the CDR1 region. TH9 also has a different H chain CDR3. The closely related JH4 or JH5 gene segments are expressed in five lupus or leprosy autoantibodies. In four of the antibodies, examples of V kappa 1, V kappa 3, or V kappa 4 and J kappa 2, or J kappa 5 segments were found. Two distinct leprosy-derived anti-DNA antibodies, 8E10 and TH3, share a completely identical V kappa sequence. This sequence differs in only two positions from that of a germ-line RF L chain gene. Several gene segments that are close to the germ line in sequence encode Ig V regions with autoantibody reactivity. These results provide a base line for determining whether these genes are precursors of more highly diversified antibodies that may be pathogenic in patients with SLE.
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PMID:The recurrent expression of variable region segments in human IgM anti-DNA autoantibodies. 249 86

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.
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PMID:An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses. 751 96

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was compared with a tissue culture assay (TC) and an enzyme immunoassay (EIA) to detect St. Louis encephalitis virus (SLEV) in mosquito pools. Overall, 1,725 mosquito pools with a low viral prevalence (3.3%-5.0%) were tested. The comparison of the EIA to TC showed that the EIA was 0.947 sensitive, 0.988 specific, and 0.987 accurate. Comparison of RT-PCR to TC showed that the RT-PCR was 0.947 sensitive, 0.980 specific, and 0.979 accurate. Comparison of the RT-PCR to EIA showed that the RT-PCR was 0.878 sensitive, 0.987 specific, and 0.982 accurate. Because of speed, accuracy, and cost, either the RT-PCR or the EIA is recommended as the primary screen. RT-PCR has an advantage over EIA, because the amplified product contains sequence information which can confirm its identity. TC, EIA, or both can be applied as a 2nd assay for the confirmation of samples suspected as positives by the RT-PCR.
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PMID:Evaluation of a reverse transcriptase-polymerase chain reaction assay for detecting St. Louis encephalitis virus using field-collected mosquitoes (Diptera: Culicidae). 890 15

Significant numbers of syncytial cells were observed in the bronchoalveolar lavage fluid (BALF) of a 42-year-old patient who had SLE with interstitial pneumonia. Electron microscopic study of the BALF cells and positive reverse transcriptase activity in the supernatant of the cultured cells revealed unknown retroviral particles in the BALF cells. No antibodies to known human retroviruses or proviral sequences were detected. Type C retroviral particles and positive reverse transcriptase activity were also observed in co-cultured U937 cells. To evaluate the pathogenic role of unknown type C retroviral particles, screening for antibodies to this retroviral particle was performed by immunofluorescence in 26 patients with idiopathic pulmonary fibrosis, 17 patients with SLE, 22 patients with lung cancer, and 58 healthy volunteers. Serum antibody to this putative type C retrovirus was detected in 24% of SLE patients, 27% of idiopathic pulmonary fibrosis patients, none of the lung cancer patients and 2% of healthy volunteers. Although no direct evidence of this virus as the pathogen for SLE could be demonstrated, a possible role in the development of SLE and interstitial pneumonia might be suggested.
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PMID:Syncytial cell formation in vivo by type C retroviral particles in the systemic lupus erythematosus (SLE) lung. 906 20

Cytokines are important regulators of lymphocyte function in SLE. However, the profile of Th1 and Th2 cytokines produced by circulating lymphocytes in human SLE has not been clearly elucidated. The aim of the present study was to characterize the gene expressions of the Th1-type cytokine IFN-gamma, and the Th2-type cytokines IL-10 and IL-4 in PBMC of 15 patients with SLE and 10 healthy individuals by a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Our results showed that expression of IFN-gamma (P = 0.0004) and IL-10 (P = 0.002) transcripts were significantly increased in PBMC of patients with SLE compared with healthy controls. By contrast, expression of IL-4 transcripts in PBMC of patients with SLE was significantly decreased compared with the healthy controls (P = 0.0008). Primary sources of IL-10 were B cells and monocytes, with variable contribution of T cells as detected in various fractions of PBMC of patients with SLE (P = 0.049). These findings support the hypothesis that the enhanced production of IFN-gamma by mononuclear cells may trigger inflammatory responses, together with the enhanced production of IL-10 resulting in autoantibody production by B cells in human SLE.
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PMID:Increased interferon-gamma (IFN-gamma), IL-10 and decreased IL-4 mRNA expression in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE). 1112 56

Female Culex tarsalis Coquillett in reproductive diapause were infected per os or by intrathoracic inoculation with western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during "fall," maintained over a simulated "winter," and then tested for virus infection and transmission in vitro and in vivo after "vernal" termination. Exposure of F1 progeny of field-collected females to cool temperatures and short daylength produced females in reproductive diapause that were reluctant to imbibe infectious virus from pledgets soaked with suspensions of virus, blood and sucrose (2.5% by volume). Those infected per os maintained virus at very low or undetectable titers. Some females that originally tested negative for WEE by plaque assay on Vero cell culture tested positive by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Vero cell culture after passage in mosquito cells. Few females became infected orally with SLE, but these infected females developed elevated titers. Females inoculated with SLE retained their infection through winter and then transmitted readily in vitro and in vivo. Feeding on a vertebrate host after diapause termination significantly increased the titer of SLE in previously infected females. These experiments simulated how infections acquired either horizontally or vertically may provide mechanisms for WEE and SLE overwintering. Attempts to detect infected females during winter following a summer with enzootic WEE activity were negative by both RT-PCR and plaque assay.
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PMID:Simulated overwintering of encephalitis viruses in diapausing female Culex tarsalis (Diptera: Culicidae). 1193 Dec 62

Three mosquito-borne human encephalitis viruses (eastern equine encephalitis virus [EEE], St. Louis encephalitis virus [SLE], and La Crosse encephalitis virus [LAC]) are sympatric in the southeastern United States. However, little is known concerning the temporal and spatial pattern of the distribution of these viruses in this area. As part of surveillance activities to detect the transmission of these 3 viruses in the Tennessee Valley area, we developed a single-tube multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay capable of detecting these 3 mosquito-borne viruses in a single reaction. Three viruses were differentiated by size of amplified products. Sensitivities of the multiplex RT-PCR assay for SLE, EEE, and LAC were 1-3 log median tissue culture infective doses per pool, roughly comparable to the reported sensitivity of PCR detection assays for the individual viruses, and 1 log more sensitive than antigen-capture assays for SLE and EEE. The sensitivity of the multiplex PCR was not changed significantly when carried out in the presence of extracts prepared from 50 uninfected mosquitoes. The cost of the assay is estimated at $2.98 per test, similar to the cost of other RT-PCR-based assays for viruses. However, adaptation of the RT-PCR to a multiplex format adds less than $0.01 to the per-unit cost of an RT-PCR assay targeting a single virus species. Analysis of these data suggests that the single-tube multiplex RT-PCR assay represents a sensitive, specific, cost-effective, and rapid method for monitoring activities of the 3 endemic mosquito-borne human encephalitis viruses in mosquito populations in the southeastern United States.
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PMID:Simultaneous detection of three mosquito-borne encephalitis viruses (eastern equine, La Crosse, and St. Louis) with a single-tube multiplex reverse transcriptase polymerase chain reaction assay. 1199 26

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.
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PMID:Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay. 1203 58


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