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

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.
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PMID:Partial sequence analysis of cloned dengue virus type 2 genome. 380 28

In 3 laboratory experiments, mosquitoes were fed hepatitis C virus (HCV)-RNA positive blood by using membrane feeders, separated into head, thorax, and abdomen, and tested by a reverse transcriptase polymerase chain reaction for HCV-RNA. HCV did not replicate or disseminate in mosquitoes that had ingested blood from patients that were HCV-viremic positive. When yellow fever mosquitoes, Aedes aegypti (L.), were held for 1, 3, 7, 14, and 21 d after feeding, HCV-RNA was detected in the abdomens of 5/5 mosquitoes at 1 d after feeding; remaining tissues were negative with the exception of a single positive head at 7 d. In agreement, HCV-RNA was detected in Asian tiger mosquito, Aedes albopictus Skuse, and Anopheles stephensi Liston abdomens at 1 d, but not 3 d after feeding no HCV-RNA was detected in heads or thoraces. In addition, HCV-RNA was detected in heads of Ae. aegypti at 10 and 20 min, but not at 30 min, after feeding. The latter results raise the possibility of HCV contamination of mouthparts and, theoretically, mechanical transmission of this virus.
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PMID:Experimental evidence against replication or dissemination of hepatitis C virus in mosquitoes (Diptera:Culicidae) using detection by reverse transcriptase polymerase chain reaction. 866 87

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).
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PMID:Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers. 915 52

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50 microliters assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, and IU of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37 degrees C for RT followed by a variable amount of cycles of two-step PCR amplification (92 degrees C for 60 sec, 53 degrees C for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 10(2.8) TCID 50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.
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PMID:A simple reverse transcription-polymerase chain reaction for dengue type 2 virus identification. 933 7

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 microliters assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37 degrees C for reverse transcription followed by 30 cycles of two-step PCR amplification (92 degrees C for 60 seconds, 53 degrees C for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10(3, 6) TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination.
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PMID:Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription-polymerase chain reaction (RT-PCR) method. 939 19

A retrotransposon named Lian-Aa1 was discovered in an intron of an AaHR3-1 gene of the yellow fever mosquito, Aedes aegypti. This retrotransposon contained a long open reading frame with 1,219 amino acids that included endonuclease, reverse transcriptase, and RNase H domains. It was shown that in the Rock strain of Ae. aegypti, there were up to 1,380 copies of Lian elements, equivalent to 0.8% of the entire genome. Five additional copies of Lian elements were isolated, mapped by restriction digestion, and partially sequenced. The 5' and 3' ends of the Lian family were determined by comparing the terminal sequences of the six copies and were subsequently confirmed by the identification of putative target duplications flanking Lian-Aa1 and Lian-Aa2. The Lian family is likely a novel family of non-long-terminal-repeat (non-LTR) retrotransposons that terminate in a repeat of (CTGA-TAC)2. On average, the six copies of Lian elements showed only 0.6% sequence divergence at the nucleotide level in both a 735-bp region at the 5' end and a 1,124-bp coding region. Genomic Southern blots also revealed a very high degree of similarity among hundreds of Lian elements, suggesting very recent activity of Lian. Furthermore, all six analyzed Lian elements were closely associated with one or more different families of repetitive elements. It is possible that these associations could reflect the complex relationship between Lian elements and the rest of the Ae. aegypti genome. Phylogenetic analyses based on the reverse transcriptase, domains of 36 non-LTR retrotransposons including Lian-Aa1 identified five major subgroups that were supported by bootstrap replications. In contrast to the majority of non-LTR retrotransposons, Lian-Aa1 has an RNase H domain that is similar to a few other non-LTR retrotransposons and some retroviruses, which is consistent with the previously proposed independent assortment of different domains during the evolution of retroelements.
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PMID:Structural, genomic, and phylogenetic analysis of Lian, a novel family of non-LTR retrotransposons in the yellow fever mosquito, Aedes aegypti. 965 85

To monitor early and late events of immune system activation after primary and secondary flavivirus infection, 17 healthy persons were vaccinated with the standard 17D vaccine virus strain of yellow fever (YF). Twelve of these persons had not received YF vaccine previously and 5 had been vaccinated once at least 10 years before. Viremia and various parameters of humoral and cellular immune activation were followed daily for 7 days and weekly thereafter. Viremia was detected by reverse transcriptase-polymerase chain reaction in all 12 first-time vaccinees beginning from the second to the sixth day after vaccination; most tested positive between the fourth and sixth day. Infectious 17D virus was detected using a plaque forming assay in the serum of 7 of the 12 first-time vaccinees. As first parameters of immune activation, neopterin and beta2-microglobulin markedly increased between day 2 and day 6 postvaccination. In parallel to the viremia, circulating CD8+ T-cells significantly increased, with peak levels at day 5 after primary vaccination, indicating an activation of the cellular immune system. Neither viremia nor significant changes of these activation markers were observed in the five revaccinated persons. Neutralizing antibodies directed against the 17D vaccine strain developed in all persons within 2 weeks after vaccination. No correlation was found between the extent of viremia and the titer of neutralizing antibodies. Revaccination was followed by a minor and transient increase of neutralizing antibodies. High titers of neutralizing antibodies persisted for at least 10 years after primary vaccination.
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PMID:Development of viremia and humoral and cellular parameters of immune activation after vaccination with yellow fever virus strain 17D: a model of human flavivirus infection. 974 73

A novel family of non-long-terminal-repeat (non-LTR) retrotransposons, named MosquI, was discovered in the yellow fever mosquito, Aedes aegypti. There were approximately 14 copies of MosquI in the A. aegypti genome. Four of the five analyzed MosquI elements were truncated at the 5' ends while one of them, MosquI-Aa2, was full-length. All five MosquI elements ended with 4-10 TAA tandem repeats, as the Drosophila I factors do. Interestingly, MosquI elements were often found near genes and other repetitive elements. The 6,623-bp MosquI-Aa2 contained two open reading frames (ORFs) flanked by a 404-bp 5' untranslated region and a 326-bp 3' untranslated region. The two ORFs code for nucleocapsids, endonuclease, reverse transcriptase, and RNase H domains. Although overall structural and sequence comparisons suggest that MosquI is highly similar to the Drosophila I factors, phylogenetic analysis based on the reverse transcriptase domains of 40 non-LTR retrotransposons indicate that MosquI and I factors are likely paralogous elements which may have been separated before the split between the ancestors of mollusca and arthropoda. Pairwise comparisons between the four truncated MosquI elements showed 96.7%-99.5% identity at the nucleotide level, while comparisons between the full-length MosquI-Aa2 and the truncated copies showed only 80.2%-81.8% identity. These comparisons and preliminary phylogenetic analyses suggest that the full-length and truncated MosquI elements may belong to two subfamilies originating from two source genes that diverged a long time ago. In contrast to the defective I factors in Drosophila melanogaster, which are likely very old components of the genome, the truncated MosquI elements seem to have been recently active. Finally, the genomic distribution and evolution of MosquI elements are analyzed in the context of other non-LTR retrotransposons in A. aegypti.
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PMID:MosquI, a novel family of mosquito retrotransposons distantly related to the Drosophila I factors, may consist of elements of more than one origin. 1060 10

We studied five fatal cases of dengue haemorrhagic fever (DHF), confirmed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method, in Vietnamese children. The liver seems to be a target for dengue virus, so postmortem examinations were performed to investigate elementary lesions, local recruitment of inflammatory cells and whether the virus was present in target cells of the liver. We detected severe, diffuse hepatitis with midzonal necrosis and steatosis in two patients, focal areas of necrosis in two patients, and normal histology in one patient. Dengue virus antigen was detected using immunohistochemistry in hepatocytes from necrotic areas in four cases. There was no recruitment of polymorphonuclear cells, and no lymphocytes were detected in the liver lesions of patients who died from DHF. Lymphocytic infiltration occurred in only one hepatitis B virus-positive patient, with no signs of chronic hepatitis. Kupffer cells had mostly been destroyed in cases with focal or severe necrosis. TUNEL tests were positive in necrotic areas, with positive cells forming clusters, suggesting that an apoptotic mechanism was involved. Thus, we suggest that the hepatocyte and Kupffer cells may be target cells supporting virus replication and that the councilman body is an apoptotic cell, as in the pathogenesis of yellow fever.
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PMID:Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children. 1125 11


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