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)

The warm climate of Israel and mishandling of the cadavers during transit to the laboratory requires an accurate method for diagnosis of rabies in decomposed tissues. By using the reverse transcriptase polymerase chain reaction (RT-PCR) 10 decomposed brain samples that collected between 1998 and 2000 were diagnosed as negative by direct fluorescent antibody test (FAT), were found positive. Three of the 10 decomposed brains were confirmed as positive by isolation of rabies virus in tissue culture and by mouse inoculation (MIT) while the other seven decomposed samples were found positive only by RT-PCR. Direct sequencing and molecular analysis of a 328bp fragment of the N gene of all the rabies sequences confirmed their geographical origin. These results demonstrated the importance of the RT-PCR in the detection of rabies virus in decomposed naturally infected brains, especially in cases when the sample is not suitable for other laboratory assays. Thus, the RT-PCR can provide a positive diagnosis; however, when a negative result is obtained due to the nature of the decomposed tissue that can be caused by technical reasons and a false negative might be the case.
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PMID:Rabies virus detection by RT-PCR in decomposed naturally infected brains. 1203 39

A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. The N gene was used as the target for the probes as it is well conserved and has been intensively used to genotype rabies isolates. Additionally, it was found to contain regions specific to each genotype conducive to probe design. The RT-PCR assay described amplifies a portion of the nucleoprotein gene of all 107 rabies and rabies-related viruses, but none of the other viruses tested. Inclusion of TaqMan-genotype-specific probes in the RT-PCR assay permits rapid identification of the virus present. By combining RT-PCR with TaqMan genotyping probes suspect rabies virus isolates can be identified in a single closed tube system that prevents potential PCR-product carry over contamination.
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PMID:A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan technology. 1217 39

Increased travel to exotic destinations around the world is escalating the risk that an emerging virus may be imported into the UK. Rabies should be considered in the differential diagnosis of any encephalitic illness presenting in an appropriate epidemiological context. Molecular diagnostic tests that can rapidly discriminate rabies from other suspected infections will influence the use of anti-rabies prophylaxis for potential contacts with the victim. In 2001, the UK had two confirmed human rabies cases, imported from the Philippines and Nigeria, respectively. In case one, hemi-nested reverse transcriptase polymerase chain reaction (hn-RT-PCR) and automated sequencing confirmed the presence of rabies virus (RABV) within both the saliva and skin specimens within 36 h of sample submission. Subsequent phylogenetic analysis using a partial sequence of the nucleoprotein (N-) gene segment demonstrated that the virus was closely related to that of canine variants currently circulating in the Philippines. In the second case, the fluorescent antibody test and reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the diagnosis on post-mortem tissue. Phylogenetic analysis of two genomic segments of this isolate confirmed that it was a classical RABV (genotype 1) of the Africa 2 subgroup. These cases have highlighted the capability of molecular diagnostic tests for the rapid identification and subsequent genotyping of RABV to host and geographical location. In the first instance, rabies diagnosis often rests on clinical and epidemiological grounds. Negative tests, even late in the illness, do not exclude the diagnosis as these tests are never optimal and are entirely dependent on the nature and quality of the sample supplied. For this reason, rapid molecular detection and virus typing will be essential in considering the appropriate medical treatment regimen for a patient. In addition, an early diagnosis may decrease the number of unnecessary contacts with the patient and reduce the requirement for invasive and costly interventions. Rabies should form part of a differential diagnosis for any patient presenting with a history of travel to a rabies endemic country and displaying an undiagnosed encephalopathy.
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PMID:Risk factors associated with travel to rabies endemic countries. 1267 34

An epidemic-geographic rabies study was carried out in which 72 animal and human brain samples were analyzed for Lyssaviruses by a direct immunofluorescent technique (DIFT) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Fifty-two samples were also tested by a mouse inoculation test. Lyssavirus RNA was detected in 60 of 72 samples. Five DIFT-negative bat samples tested by a nested PCR assay showed evidence of the presence of rabies virus RNA. Sequencing of amplified rabies virus nucleoprotein encoding segments of a selection of the samples resulted in the formation of clusters, corresponding to samples originating from cattle and equines from the same hydrographic basin. Genomically related Lyssavirus strains of bat origin were found in each cluster, most likely because of the role of the bat in the epidemiology of the virus. All samples studied were of genotype 1. With exception of the human sample, all were distinct from the reference sample.
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PMID:Study of lyssaviruses of bat origin as a source of rabies for other animal species in the State of Rio De Janeiro, Brazil. 1293 3

A 55-year-old bat conservationist was admitted to Ninewells Hospital, Dundee, Scotland, on November 11, 2002, with an acute haematemesis. He gave a 5-day history of pain and paraesthesia in the left arm, followed by increasing weakness of his limbs with evidence of an evolving encephalitis with cerebellar involvement. The patient had never been vaccinated against rabies and did not receive postexposure treatment. Using a hemi-nested reverse transcriptase-polymerase chain reaction (RT-PCR), saliva samples taken intravitam from different dates proved positive for rabies. A 400-bp region of the nucleoprotein gene was sequenced for confirmation and identified a strain of European bat lyssavirus (EBLV) type 2a. The diagnosis was confirmed using the fluorescent antibody test (FAT) and by RT-PCR on three brain samples (cerebellum, medulla, and hippocampus) taken at autopsy. In addition, a mouse inoculation test (MIT) was performed. Between 13 and 17 days postinfection, clinical signs of a rabies-like illness had developed in all five inoculated mice. Brain smears from each infected animal were positive by the FAT and viable virus was isolated. This fatal incident is only the second confirmed case of an EBLV type-2 infection in a human after exposure to bats.
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PMID:Case report: isolation of a European bat lyssavirus type 2a from a fatal human case of rabies encephalitis. 1293 4

Recombinant rabies virus (RV) vaccine strain-based vectors have been successfully developed as vaccines against other viral diseases (J. P. McGettigan et al., J. Virol. 75:4430-4434, 2001; McGettigan et al., J. Virol. 75:8724-8732, 2001; C. A. Siler et al., Virology 292:24-34, 2002), and safety concerns have recently been addressed (McGettigan et al., J. Virol. 77:237-244, 2003). However, size limitations of the vectors may restrict their use for development of vaccine applications that require the expression of large and multiple foreign antigens. Here we describe a new RV-based vaccine vehicle expressing 4.4 kb of the human immunodeficiency virus type 1 (HIV-1) Gag-Pol precursor Pr160. Our results indicate that Pr160 is expressed and processed, as demonstrated by immunostaining and Western blotting. Electron microscopy studies showed both immature and mature HIV-1 virus-like particles (VLPs), indicating that the expressed HIV-1 Gag Pr55 precursor was processed properly by the HIV-1 protease. A functional assay also confirmed the cleavage and functional expression of the HIV-1 reverse transcriptase (RT) from the modified RV genome. In the next step, we constructed and recovered a new RV vaccine strain-based vector expressing a chimeric HIV-1(89.6P) RV envelope protein from an additional RV transcription unit located between the RV nucleoprotein (N) and phosphoprotein (P) in addition to HIV-1 Pr160. The 2.2-kb chimeric HIV-1/RV envelope protein is composed of the HIV-1 Env ectodomain (ED) and transmembrane domain (TD) fused to RV glycoprotein (G) cytoplasmic domain (CD), which is required for efficient incorporation of HIV-1 Env into RV particles. Of note, the expression of both HIV-1 Env and HIV-1 Pr160 resulted in an increase in the rhabdoviral genome of >55%. Both rhabdovirus-expressed HIV-1 precursor proteins were functional, as indicated by RT activity and Env-based fusion assays. These findings demonstrate that both multiple and very large foreign genes can be effectively expressed by RV-based vectors. This research opens up the possibility for the further improvement of rhabdovirus-based HIV-1 vaccines and their use to express large foreign proteins, perhaps from multiple human pathogens.
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PMID:Functional human immunodeficiency virus type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and env expressed from a single rhabdovirus-based vaccine vector genome. 1451 39

Five hundred rodents and shrews (Rattus norvegicus: 458, Rattus rattus: 28, Rattus exulans: 5, Mus musculus: 4 and Suncus murine: 5) trapped from the fresh food markets around Bangkok area were investigated for rabies virus and Hantaan virus infections. No rabies viral antigens in the animals' brains were detected by direct immunofluorescence. On the other hand, antibodies to Hantaan virus were demonstrated in the sera of 7 (1.53%) R. norvegicus caught in various markets using a particle agglutination technique. Further determination of the viral genome in rat lung tissue was performed by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR, 3 (0.66%) out of 7 were positive. HindIII and HifI restriction enzyme analyses showed the pattern of the Hantaan virus genome in 2 samples and that of the Seoul virus genome in the other. The results of the present study suggest that rodents from Bangkok's fresh food markets did not carry rabies. Thus, getting rid of rabies in dogs or cats in the Bangkok area may be easier than anticipated because there are no sources of asymptomatic reservoirs. This may result in the low incidence of rabies patients observed in Bangkok. On the contrary, the presence of antibodies and the Hantaan virus genome and Seoul virus genome in R. norvegicus will definitely provide evidence for physicians to be aware of hemorrhagic fever with renal syndrome (HFRS) and other clinical settings of Hantaan/Seoul virus disease in patients with a history of having contact with rats or their excreta.
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PMID:Prevalence of rabies virus and Hantaan virus infections in commensal rodents and shrews trapped in Bangkok. 1469 82

A simplified hemi-nested reverse transcriptase polymerase chain reaction (hnRT-PCR) has been developed to determine specifically the European Bat Lyssavirus 1 (EBLV-1) nucleoprotein gene. The specificity of this method was determined by using the seven genotypes of lyssavirus by RT-PCR, Southern blot and sequence analysis. Compared to the rabies diagnostic methods, the hnRT-PCR showed a higher sensitivity for the detection of small amounts of EBLV-1 virus. In view of these results, we suggest this new hnRT-PCR should be performed for the epidemiological survey of bat colonies, also providing rapid detection and genotyping of EBLV-1 until now encountered in all naturally infected bats in France.
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PMID:Development of a hemi-nested RT-PCR method for the specific determination of European Bat Lyssavirus 1. Comparison with other rabies diagnostic methods. 1512 4

The challenge virus standard (CVS) strain and a wild isolate from a Mexican child who died of hematophagous bat (Desmodus rotundus)-transmitted rabies were injected intracerebrally into BALB/c mice. Brains obtained from infected mice were immersed in 80%, 50%, and 40% glycerine/phosphate-buffered saline (PBS). RNA was extracted from brains on days 1, 2, 3, 7, 21, and 60, and reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) tests were performed for rabies virus characterization. Storage temperature variation was recorded during the preservation period. The RT-PCR-RFLP tests were successfully performed on brain samples preserved in 50% glycerine/PBS, but not in those preserved in 80% or 40% glycerine/PBS. Temperatures ranged from 12 to 33 degrees C and were not harmful, provided that 50% glycerine/PBS was used. We concluded that brain samples obtained and stored under field conditions (i.e. without refrigeration) for up to 60 d can arrive at a reference laboratory in an adequate condition for viral RNA analysis.
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PMID:Preservation of rabies virus RNA from brain tissue using glycerine. 1530 22

The purposes of this study were to elucidate the role of cytokine upregulation in the pathogenesis of rabies encephalitis and to compare the detection of Negri bodies with that of rabies protein by immunohistochemistry and rabies RNA by reverse transcriptase (RT) in situ PCR for its diagnosis. Negri bodies were evident in 4/7 of the documented rabies cases; viral protein and viral RNA were detected in each case. The average number of rabies-infected cells, determined by counting 150 neurons in serial sections in areas where viral protein was evident, with the three different detection methods was: Negri bodies (<1/150), immunohistochemistry (4/150), and RT in situ PCR (49/150). No rabies protein or RNA was detected in four control brain tissues that were read with the rabies cases in a blinded fashion. The ratio of cells expressing tumor necrosis factor alpha (TNFalpha) or inducible nitric oxide synthetase (iNOS) to 1 SSI-1/SOCS-1 (suppressors of cytokine signaling) expression, which is a novel class of negative feedback regulators of cytokine receptor signaling, was markedly increased only in the areas where many viral infected cells were present. Colabeling experiments showed that most of the cells expressing iNOS or TNFalpha were not virally infected, but rather adjacent to rabies-infected neurons. We conclude that RT in situ PCR for rabies virus is the most accurate test for the determination of viral load in rabies encephalitis. Further, the disease is characterized by massive viral infection of neurons in a markedly focal distribution in conjunction with a concomitant upregulation of cytokine expression in adjacent, noninfected cells that may be due, in part, to SOCS downregulation.
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PMID:Molecular detection of rabies encephalitis and correlation with cytokine expression. 1538 58


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