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)

Necropsy of an older dog submitted for evaluation of renal and central nervous system disease revealed histologic lesions compatible with West Nile viral encephalitis and myocarditis, as seen in other species. Using reverse transcriptase-polymerase chain reaction detection of envelope sequences, viral RNA was detected in most organs, and quantitative polymerase chain reaction revealed that at least 1,000 times more RNA was present in kidney than in brain, heart, spleen, or lung. Immunohistochemical evaluation of the kidney revealed intense staining of West Nile viral antigens in renal tubular epithelium and casts located within multifocal granulomatous interstitial inflammation. A canine immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay was developed, and patient serum was strongly positive for viral antibody. Retrospective and ongoing evaluation of sera from dogs with neurological disease and of those submitted for heartworm testing detected 4 dogs that were subclinically infected but without additional sickness. Judged by this experience, the kidney of West Nile virus-infected dogs may be an important target organ, one that might be suitable for antemortem biopsy. The presence of virus-specific IgM was demonstrated in the serum of this dog, and finding 4 positives among 169 additional canine sera received since late July 2002 suggests that seroconversion appears to be relatively uncommon in dogs during the outbreak in Missouri.
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PMID:Serological, reverse transcriptase-polymerase chain reaction, and immunohistochemical detection of West Nile virus in a clinically affected dog. 1291 12

We have developed a reporting replicon of West Nile virus (WNV) that could be used to quantitatively distinguish viral translation and RNA replication. A Renilla luciferase (Rluc) gene was fused in-frame with the open reading frame of a subgenomic replicon in the position where the viral structural region was deleted, resulting in RlucRep. Transfection of BHK cells with RlucRep RNA yielded two distinctive Rluc signal peaks, one between 2 and 10 h and the other after 26 h posttransfection. By contrast, only the 2- to 10-h Rluc signal peak was observed in cells transfected with a mutant replicon containing an inactivated viral polymerase NS5 (RlucRep-NS5mt). Immunofluorescence and real-time reverse transcriptase PCR assays showed that the levels of viral protein expression and RNA replication increased in cells transfected with the RlucRep but not in those transfected with the RlucRep-NS5mt. These results suggest that the Rluc signal that occurred at 2 to 10 h posttransfection reflects viral translation of the input replicon, while the Rluc activity after 26 h posttransfection represents RNA replication. Using this system, we showed that mutations of conserved sequence (CS) elements within the 3' untranslated region of the mosquito-borne flaviviruses did not significantly affect WNV translation but severely diminished or completely abolished RNA replication. Mutations of CS1 that blocked the potential base pairing with a conserved sequence in the 5' region of the capsid gene (5'CS) abolished RNA replication. Restoration of the 5'CS-CS1 interaction rescued viral replication. Replicons containing individual deletions of CS2, repeated CS2 (RCS2), CS3, or RCS3 were viable, but their RNA replication was dramatically compromised. These results demonstrate that genome cyclization through the 5'CS-CS1 interaction is essential for WNV RNA replication, whereas CS2, RCS2, CS3, and RCS3 facilitate, but are dispensable for, WNV replication.
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PMID:Functional analysis of mosquito-borne flavivirus conserved sequence elements within 3' untranslated region of West Nile virus by use of a reporting replicon that differentiates between viral translation and RNA replication. 1294 11

Since the initial outbreak of West Nile virus (WNV) in the northeastern United States in 1999, the virus has rapidly spread westward and southward across the USA, causing high mortality in crows as well as sporadic mortality in horses, humans, and a wide variety of birds. In 2002 the epidemic widened as hundreds of equine and human cases and sporadic cases in other mammalian species were reported. This is the first report of WNV infection in three Eastern fox squirrels (Sciurus niger). Neurologic signs included head tilt, uncoordinated movement, paralysis, and tremors. Gross lesions were absent. Microscopic lesions consisted of lymphoplasmacytic inflammation involving the brain, heart, kidney, and liver. Formalin-fixed tissues from the three squirrels were tested for WNV antigen by immunohistochemical staining and for WNV-specific RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The kidneys of all three squirrels stained positive with immunohistochemistry for WNV, whereas the brain and heart were positive in only one animal. Two of the three squirrels were positive for WNV by RT-PCR.
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PMID:West Nile virus infection in Eastern fox squirrels (Sciurus niger). 1460 26

This study reports on the development of a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific detection of turkey coronavirus (TCoV). Of the several sets of primers tested, 1 set of primers derived from the P gene and 2 sets derived from the N gene of TCoV could amplify the TCoV genome in the infected samples. The RT-PCR was sensitive and specific for TCoV and did not amplify other avian RNA and DNA viruses tested except the infectious bronchitis virus (IBV). To overcome the problem of IBV amplification, a set of separate primers was designed from the spike protein gene of IBV. The RT-PCR under the same conditions as above could effectively differentiate between TCoV and IBV. The closely related bovine coronavirus and transmissible gastroenteritis virus of pigs were differentiated from TCoV using the same RT-PCR with slight modifications. The results of RT-PCR correlated well with the results of the immunofluorescent test for the same samples tested at the Purdue University Animal Disease Laboratory, West Lafayette, Indiana. The nucleotide sequence and projected amino acid sequence comparison of the P gene of different isolates of TCoV from 5 different states in the United States revealed a close association among the different isolates of TCoV.
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PMID:A reverse transcriptase-polymerase chain reaction assay for the diagnosis of turkey coronavirus infection. 1466 27

West Nile virus (WNV) is an important arthropod borne flavivirus; usually causes a mild infection called West Nile fever (WNF) in human and horses. Mosquitoes are the principal vectors of WNV. Various Culex species are found to act as vectors in different geographical regions. The virus is maintained in a bird-mosquito cycle in nature. In India, Culex mosquitoes are tentatively incriminated as vectors of WNV. Experimental studies have shown that Culex tritaeniorhynchus, Cx. vishnui, Cx. bitaeniorhynchus and Cx. univittatus, Culex pipiens fatigans and Aedes albopictus could act as potential vectors of WNV. Transovarial transmission of WNV has been experimentally demonstrated in Culex mosquitoes. Apart from mosquitoes, the role of other arthropods is also considered in the maintenance of WNV during inter-enzootic periods. The possible role of ardeid birds in the maintenance of WNV has been described in India. Though very few clinically overt cases of human encephalitis due to WNV are observed, Japanese encephalitis virus (JEV) is found to dominate in southern India. WNF in horses has not been documented in India. JEV immunized monkeys were protected from WNV challenge and the WNV immunization was found to reduce the disease severity due to JEV. Based on the limited genome sequence analysis, the Indian isolates are grouped together under the genetic lineage-I. WNV infection is diagnosed by IgM antibody capture enzyme linked immunosorbant assay, haemagglutination inhibition test, neutralization test and reverse transcriptase-polymerase chain reaction (RT-PCR). For the effective control of Culex mosquitoes, integrated vector control strategies are recommended. Specific methods are not available for the treatment of WNV infection. However, in patients with encephalitis supportive therapy is recommended. Though a few candidate vaccines are under laboratory trial, no vaccine has been available commercially for the control of WNV infection in human and animals. In view of the global interest on WNV, this paper describes the present status of WNV in India.
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PMID:West Nile virus: the Indian scenario. 1470 Mar 42

VecTest assays for detecting eastern equine encephalitis virus (EEE) and western equine encephalitis virus (WEE) antigen in mosquito pools were evaluated to determine their sensitivity and specificity by using a range of EEE, WEE, St. Louis encephalitis virus (SLE), and West Nile virus (WN) dilutions as well as individual and pooled mosquitoes containing EEE or WEE. The EEE test produced reliable positive results with samples containing > or = 5.3 log10 plaque-forming units (PFU) of EEE/ml, and the WEE test produced reliable positive results with samples containing > or = 4.7 log10 PFU WEE/ml. Both assays detected the respective viral antigens in single virus-positive mosquitoes and in pools containing a single positive mosquito and 49 negative specimens. The SLE and WN assays also contained on the dipsticks accurately detected their respective viruses. No evidence was found of cross reaction or false positives in any of the tests. The VecTest assays were less sensitive than the EEE- and WEE-specific TaqMan reverse transcriptase polymerase chain reaction and Vero cell plaque assay, but appear to be useful for detecting arboviruses in mosquito-based arbovirus surveillance programs.
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PMID:Sensitivity of the VecTest antigen assay for eastern equine encephalitis and western equine encephalitis viruses. 1471 Jul 52

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63 degrees C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 10(4) PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.
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PMID:Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. 1471 62

The North American West Nile virus (WNV) epizootic, which began in 1999, has caused significant morbidity and mortality in horses. Because experimental infection has failed to consistently produce encephalitis in inoculated horses, investigation of naturally occurring cases was used to optimize strategies for diagnosis of this disease. Although WNV RNA could be detected by reverse transcriptase-polymerase chain reaction (RT-PCR) performed on whole blood collected from both clinically affected horses and unaffected herdmates, the diagnostic sensitivity of this approach was low compared with IgM-capture enzyme-linked immunosorbent assay. In addition, it was observed that 18.5% of herdmates of clinically ill horses seroconverted to WNV yet exhibited no overt clinical signs of WNV encephalitis. West Nile viral RNA was detected in neural tissue of 46 of 64 dead horses that were suspected of having WNV encephalitis. Some of these animals were IgM negative or had not been tested serologically. A primary cause of death other than WNV encephalitis was identified in 15 of the 64 cases, whereas the final diagnosis for 3 of these cases remains unresolved. Quantitative RT-PCR analysis of neural tissue from WNV RNA-positive horses demonstrated that the medulla contained the highest mean concentration of viral RNA and that WNV RNA could be detected in samples extracted from formalin-fixed neural tissue. A comparison of WNV RT-PCR amplification strategies found that nested RT-PCR improved diagnostic sensitivity only slightly over a single round of amplification and that a quantitative (TaqMan) assay had sensitivity and specificity that were equivalent to those of nested amplification.
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PMID:Diagnosis of West Nile virus infection in horses. 1497 40

This article describes the methods used to investigate 407 outbreaks of acute non-bacterial gastroenteritis occurring in the North-West of England between January 2000 and July 2001 and suspected to be caused by noroviruses (NV) [Mayo (2002) Arch Virol 147:1655-1663]. These included 319 outbreaks in hospitals and nursing homes and 88 other settings. Eight hundred and seventy-one faecal samples from 407 outbreaks were tested using electron microscopy (EM), an enzyme-linked immunosorbent assay (ELISA) specific for Grimsby virus (GRV) capsid antigen and/or by reverse transcriptase-polymerase chain reaction (RT-PCR) for NV, allowing the utility of each assay for routine diagnosis to be assessed. Preliminary genomic characterisation of detected strains was performed using the heteroduplex mobility assay (HMA) and DNA sequencing. The results demonstrate the continuing predominance of GII-4 GRV strain of NV as a cause of outbreaks, particularly in hospital and nursing home settings. Overall, NV were detected in 223/407 (55%) of outbreaks tested. However, a wide range of apparently diverse strains was identified, including several not previously reported. Genomic characterisation revealed clusters of linked outbreaks not recognised previously.
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PMID:Methods for the detection and characterisation of noroviruses associated with outbreaks of gastroenteritis: outbreaks occurring in the north-west of England during two norovirus seasons. 1512 5

During 2002, West Nile virus (WNV) infection was diagnosed in 11 birds housed in outdoor exhibits at 5 zoological institutions in Kansas. Eight birds were examined because of neurologic abnormalities; 2 died suddenly without any clinical signs of disease. Results of CBCs and serum biochemical testing were nonspecific. Results of a plaque reduction neutralization test to detect circulating antibodies against WNV were positive for 2 of 8 birds. Results of a reverse transcriptase-polymerase chain reaction assay of an oral cavity swab specimen for WNV RNA were positive for 4 of 5 birds. One bird survived; the remaining 10 died or were euthanatized, with 9 of the 10 dying or being euthanatized within 3 days of the onset of clinical signs. In all 10 birds that died or were euthanatized, WNV infection was confirmed on postmortem examination by means of specific testing. Findings in these birds suggest that West Nile virus infection can be difficult to diagnose antemortem because clinical signs mimic those associated with other more common avian diseases. Neither of the antemortem diagnostic tests was definitive for diagnosing WNV infection in these cases.
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PMID:Clinical signs and results of specific diagnostic testing among captive birds housed at zoological institutions and infected with West Nile virus. 1515 35


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