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 polyadenylation of Fowl Plague Viral RNA and of Influenza A/Victoria Viral RNA using E. coli poly (A) polymerase and the subsequent reverse transcription of the polyadenylated species is reported. We have shown that all 8 genome fragments are adenylated and that an average of 25--30 adenylic acid residues per molecule is sufficient for maximal transcription with reverse transcriptase. The cDNA product is 95% sensitive to Sl-nuclease and hybridisation analysis against viral RNA reveals it to be a faithful copy of the RNA. Amongst the transcription products are long, discrete copies of genes 1--8, the lengths of which are comparable with those of the vRNA determined by electrophoresis on formamide acrylamide gels. These single-stranded cDNAs have been further transcribed to form double-stranded products with hair-pin structures at one end. Analysis of this material on native acrylamide gels revealed some DNA bands corresponding to the predicted sizes for genes 4--8.
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PMID:Polyadenylation and reverse transcription of influenza viral RNA. 8 38

A reverse transcriptase PCR (RT-PCR) was used for rapid determination of the hemagglutinin (HA) cleavage site sequence, a marker for the virulence potential of avian influenza viruses. When applied to specimens from chickens experimentally infected with either a virulent or an avirulent virus, RT-PCR uniformly detected the HA gene, even in specimens that were negative for virus by standard testing in eggs. This technique, combined with sequencing of the HA cleavage site, offers a rapid and sensitive way to assess the virulence potential of avian influenza viruses. Early detection of field isolates with virulence-associated structural motifs at the HA cleavage site would allow better control of influenza among large poultry populations.
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PMID:Direct reverse transcriptase PCR to determine virulence potential of influenza A viruses in birds. 775 90

Recent isolations of H5N2 subtype avian influenza (AI) viruses in North America have raised questions concerning their origin, transmission to commercial poultry, and potential for virulence. One ratite-origin isolate of low pathogenicity, A/emu/TX/39924/93 (H5N2), was subjected to a procedure that rapidly selects and/or amplifies highly pathogenic (HP) strains. The resulting highly virulent derivative had an altered hemagglutinin (HA) gene containing an additional six nucleotides at position 970-975 in the HA1 coding region. This resulted in an arg-lys insertion near the proteolytic cleavage site of the HA protein. The remainder of the HA sequence differed by an additional seven amino acids from the parent. The HA precursor of the derivative, but not the parent, was readily cleaved during replication in cell culture without addition of trypsin. In experimentally infected chickens, the derivative produced lesions typical of highly pathogenic avian influenza. A reverse transcriptase-polymerase chain reaction (RT-PCR) primer set was designed to amplify exclusively from molecules with the inserted six nucleotides. The set yielded product only from the selected derivative samples and not the parent. Thus, the levels of the HP variants in the parent stock were undetectable, or the insertion occurred rapidly during the selection process.
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PMID:An Arg-Lys insertion at the hemagglutinin cleavage site of an H5N2 avian influenza isolate. 887 23

An enteric disease of young turkeys, referred to as stunting syndrome (SS), causes reduced growth and impaired feed efficiency. A recently isolated virus, stunting syndrome agent, (SSA) has been found to be the etiologic agent of SS. The objective of the present study was to determine relatedness of the SSA with other viral agents. Serologic (viral neutralization and enzyme-linked immunosorbent assay [ELISA]) assays and a reverse transcriptase-polymerase chain reaction (RT-PCR) were used. The antisera against turkey enteric coronavirus (bluecomb agent), bovine coronavirus (BCV), bovine Breda-1 virus, bovine Breda-2 virus, avian infectious bronchitis virus (IBV), avian influenza virus, Newcastle disease virus (NDV), and transmissible gastroenteritis virus (TGEV) of swine were evaluated by dot-immunobinding avidin-biotin-enhanced ELISA and did not react with SSA. The homologous (anti-SSA) antiserum was positive by ELISA. Similarly, anti-SSA antiserum did not react when NDV, IBV, BCV, or TGEV was used as antigen but did react with the homologous (SSA) virus. The virus neutralization assay was performed by inoculating 24-to-25-day-old turkey embryos via the amniotic route and by assessing the embryo infectivity on the basis of gross intestinal lesions and intestinal maltase activity at 72 hr postinoculation. None of the aforementioned antisera neutralized SSA infectivity in embryos except for the homologous anti-SSA antiserum. A RT-PCR was performed with known primers specific for NDV, IBV, BCV, and TGEV. The known primers failed to amplify SSA genome but amplified their respective viral genomes. We concluded that the SSA was distinct from the viral agents that were evaluated.
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PMID:Characterization of the stunting syndrome agent: relatedness to known viruses. 1073 43

The nucleocapsid (N) gene of turkey coronavirus (TCV) was amplified by reverse transcriptase-polymerase chain reaction, cloned, and expressed in the baculovirus expression system. A recombinant baculovirus containing the TCV N gene (rBTCV/N) was identified by polymerase chain reaction and expression of TCV N protein as determined by western immunoblot analysis. Two TCV-specific proteins, 52 and 43 kDa, were expressed by rBTCV/N; one of these proteins, p52, was comparable in size to native TCV N protein. Baculovirus-expressed N proteins were used as antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for detection of TCV-specific antibodies. The ELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related avian coronavirus, but did not detect antibodies specific for other avian viruses (avian influenza, avian reovirus, avian paramyxovirus 3, avian adenovirus 1, or Newcastle disease virus). These findings indicate that baculovirus-expressed TCV N protein is a suitable source of antigen for ELISA-based detection of TCV-specific antibodies in turkeys.
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PMID:Baculovirus expression of turkey coronavirus nucleocapsid protein. 1133 74

Highly pathogenic avian influenza (HPAI) in poultry causes high morbidity and mortality, and it is a List A disease of the Office International des Epizooties. An outbreak of HPAI in commercial poultry not only causes direct disease losses but often results in trade restrictions for the affected country. Because HPAI viruses can mutate from H5 and H7 low pathogenic avian influenza viruses, it is necessary to monitor and control even the low pathogenic form of the virus. We report a practical approach for screening large numbers of isolates that uses amplification by reverse transcriptase-polymerase chain reaction of a segment of the hemagglutinin (HA) gene (536-560 bp) of H7 avian influenza viruses followed by the heteroduplex mobility assay (HMA). The HMA test compares the amplified polymerase chain reaction product from unknown samples with reference isolates, which allows the identification of new variants. The HMA test results were compared with sequence analysis of the isolates used in the study. On the basis of the HMA, we could identify several new variant viruses present in the live bird markets in the northeastern United States. New strains gave a distinct pattern of bands in the gels in accordance with the different heteroduplexes formed when their HA region amplification products were incubated together with the same amplification product of a reference strain. These differences correlate with phylogenetic analysis from sequence data.
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PMID:Heteroduplex mobility assay for detection of new avian influenza virus variants. 1206 49

A real-time reverse transcriptase PCR (RRT-PCR) assay based on the avian influenza virus matrix gene was developed for the rapid detection of type A influenza virus. Additionally, H5 and H7 hemagglutinin subtype-specific probe sets were developed based on North American avian influenza virus sequences. The RRT-PCR assay utilizes a one-step RT-PCR protocol and fluorogenic hydrolysis type probes. The matrix gene RRT-PCR assay has a detection limit of 10 fg or approximately 1,000 copies of target RNA and can detect 0.1 50% egg infective dose of virus. The H5- and H7-specific probe sets each have a detection limit of 100 fg of target RNA or approximately 10(3) to 10(4) gene copies. The sensitivity and specificity of the real-time PCR assay were directly compared with those of the current standard for detection of influenza virus: virus isolation (VI) in embryonated chicken eggs and hemagglutinin subtyping by hemagglutination inhibition (HI) assay. The comparison was performed with 1,550 tracheal and cloacal swabs from various avian species and environmental swabs obtained from live-bird markets in New York and New Jersey. Influenza virus-specific RRT-PCR results correlated with VI results for 89% of the samples. The remaining samples were positive with only one detection method. Overall the sensitivity and specificity of the H7- and H5-specific RRT-PCR were similar to those of VI and HI.
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PMID:Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. 1220 62

In 2001, all 109 retail live-bird markets (LBMs) in New York and New Jersey were surveyed for the presence of avian influenza virus (AIV) by a real time reverse transcriptase/polymer chain reaction assay (RRT/PCR) and results compared to virus isolation (VI) in embryonating chicken eggs. The RRT/PCR had a 91.9% sensitivity and 97.9% specificity in detecting presence of AIV at the market level. However, the sensitivity at the sample level is 65.87%. The RRT/PCR is a reliable method to identify AIV at the market level. In addition, a cross-sectional epidemiologic study of the LBMs showed that, during the past 12 months, markets that were open 7 days per week and those that also sold rabbits had the highest risk for being positive for AIV. Markets that were closed one or more days per week and those that performed daily cleaning and disinfecting had the lowest risk for being AIV positive.
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PMID:Epidemiologic and surveillance studies on avian influenza in live-bird markets in New York and New Jersey, 2001. 1457

A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard.
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PMID:RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza. 1457 14

A real-time reverse transcriptase/polymerase chain reaction (RRT-PCR) assay was developed using hydrolysis probes for the detection of avian influenza virus (AIV) and the H5 and H7 subtypes. The AIV specific primers and probes were directed to regions of the AIV matrix gene that are conserved among most type A influenza viruses. The H5 and H7 primers and probes are directed to H5 and H7 hemagglutinin gene regions that are conserved among North American avian influenza viruses. The sensitivity and specificity of this RRT-PCR assay was compared to virus isolation (VI) in chicken embryos with 1550 clinical swab samples from 109 live-bird markets (LBMs) in New York and New Jersey. RRT-PCR detected influenza in samples from 61 of 65 (93.8%) of the LBMs that were the sources of VI positive samples. Of the 58 markets that were positive for H7 influenza by hemagglutination inhibition assay, RRT-PCR detected H7 influenza in 56 markets (96.5%). Too few H5 positive samples were obtained to validate the H5 RRT-PCR assay in this study. Although RRT-PCR was less sensitive than VI on an individual sample basis, this study demonstrated that the AIV and H7 RRT-PCR assays are good tools for the rapid screening of flocks and LBMs.
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PMID:Development of real-time RT-PCR for the detection of avian influenza virus. 1457 15


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