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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 35 min at 62.5 degrees C. In the present study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H5N1 highly pathogenic avian influenza (HPAI). The sensitivity of the system was 0.1-0.01 plaque-forming units per reaction for HPAI-H5N1 viruses belonging to the genetically and antigenically distinct clade 1, represented by A/Vietnam/JP1203/2004, and clade 2, represented by A/Indonesia/JP283/2006. This RT-LAMP sensitivity is 10-fold higher than the sensitivity of standard one-step RT-PCR. By using viral RNAs extracted from avian influenza viruses of H1-H15 hemagglutinin (HA) subtypes and human pathogenic respiratory viruses, it was confirmed that the RT-LAMP system amplifies specifically RNA of the H5 subtype virus. The system detected H5-HA genes in throat swabs collected from humans as well as from wild birds. These results suggest that the present RT-LAMP system is a useful diagnostic tool for surveillance of recent outbreaks of the HPAI-H5N1 virus.
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PMID:Rapid diagnosis of H5N1 avian influenza virus infection by newly developed influenza H5 hemagglutinin gene-specific loop-mediated isothermal amplification method. 1721 21

Two highly pathogenic avian influenza (HPAI) virus clones that met the criteria for high-pathogenicity avian influenza viruses, by possessing a multibasic hemagglutinin (HA) cleavage site, were isolated from an H5N1 outbreak in Norfolk, England, in 1991-92. These two isolates, A/turkey/England/50-92/91 (50-92) and A/turkey/England/87-92/91 (87-92), displayed differences in virulence as determined by intravenous pathogenicity index-3 and -0, respectively. DNA sequencing of these two isolates identified 10 amino acid differences throughout the genome: three in HA and polymerase B2 (PB2) and two in polymerase B1 (PB1) and single mutations in nucleoprotein (NP) and polymerase A (PA). Serial intracerebral passages were performed in 1- or 2-day-old specific pathogen free (SPF) chicks with 87-92. Viruses reisolated from each bird passage displayed increases in intracerebral pathogenicity index values (from 0 to 1.9) and therefore virulence. Reverse transcriptase polymerase chain reaction and DNA sequencing on viruses isolated at each passage displayed nine out of the 10 mutations associated with the higher pathogenic genotype of 50-92, except for the mutation found in NP, which retained the amino acid residue associated with 87-92. Serial passage through 9-day-old SPF embryonated chicken eggs and serial intravenous passage in 6-wk-old birds could not reproduce these results. These results further highlight that nucleotide changes in the genome other than at the HA cleavage site can attenuate the virulence of HPAI viruses.
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PMID:Induced increase in virulence of low virulence highly [corrected] pathogenic avian influenza by serial intracerebral passage in chickens. 1749 93

Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures (T(m)) discriminations. Three melting peaks generated simultaneously at temperatures 85.2+/-1.0, 81.9+/-0.9 and 78.7+/-0.9 degrees C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10-100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.
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PMID:Development of a multiplex real-time PCR assay using SYBR Green 1 chemistry for simultaneous detection and subtyping of H9N2 influenza virus type A. 1751 62

The currently circulating H3N2 and H1N1 subtypes of influenza A virus cause a transient, febrile upper respiratory illness in most adults and children ("seasonal influenza"), but infants, the elderly, immunodeficient and chronically ill persons may develop life-threatening primary viral pneumonia or complications such as bacterial pneumonia. By contrast, avian influenza viruses such as the H5N1 virus that recently emerged in Southeast Asia can cause severe disease when transferred from domestic poultry to previously healthy people ("avian influenza"). Most H5N1 patients present with fever, cough and shortness of breath that progress rapidly to adult respiratory distress syndrome. In seasonal influenza, viral replication remains confined to the respiratory tract, but limited studies indicate that H5N1 infections are characterized by systemic viral dissemination, high cytokine levels and multiorgan failure. Gastrointestinal infection and encephalitis also occur. The licensed anti-influenza drugs (the M2 ion channel blockers, amantadine and rimantadine, and the neuraminidase inhibitors, oseltamivir and zanamivir) are beneficial for uncomplicated seasonal influenza, but appropriate dosing regimens for severe seasonal or H5N1 viral infections have not been defined. Treatment options may be limited by the rapid emergence of drug-resistant viruses. Ribavirin has also been used to a limited extent to treat influenza. This article reviews licensed drugs and treatments under development, including high-dose oseltamivir; parenterally administered neuraminidase inhibitors, peramivir and zanamivir; dimeric forms of zanamivir; the RNA polymerase inhibitor T-705; a ribavirin prodrug, viramidine; polyvalent and monoclonal antibodies; and combination therapies.
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PMID:Current and future antiviral therapy of severe seasonal and avian influenza. 1832 78

The objectives of our study were to determine prevalence of avian influenza viruses (AIV) on wintering grounds on the Texas Gulf Coast, USA, and to compare real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) and virus isolation for detection of AIV in cloacal swabs from wild waterfowl. Cloacal swabs were collected from hunter-harvested waterfowl from November 2005 to January 2006 at four wildlife management areas. Seven AIV were isolated from four species of ducks: Green-winged Teal (Anas crecca) in November; Blue-winged Teal (Anas discors) in November; Mottled Duck (Anas fulvigula) in December, and Northern Shoveler (Anas clypeata) in January. Prevalence of AIV for each of these species during the sampling period was 1.4, 2, 6, and 0.6%, respectively. The AIV subtypes detected were H1N2, H1N4, H4N6, H6N2, and H10N7, all previously reported in North American waterfowl. Our study identified AIV subtypes not previously reported on the Texas Gulf Coast and provides baseline data for a multiyear surveillance project.
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PMID:Avian influenza surveillance in hunter-harvested waterfowl from the Gulf Coast of Texas (November 2005-January 2006). 1843 75

Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.
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PMID:Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus. 1859 27

Influenza A virus is a major human and animal pathogen with the potential to cause catastrophic loss of life. The virus reproduces rapidly, mutates frequently and occasionally crosses species barriers. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments. Here we demonstrate the importance to viral replication of a subunit interface in the viral RNA polymerase, thereby providing a new set of potential drug binding sites entirely independent of surface antigen type. No current medication targets this heterotrimeric polymerase complex. All three subunits, PB1, PB2 and PA, are required for both transcription and replication. PB1 carries the polymerase active site, PB2 includes the capped-RNA recognition domain, and PA is involved in assembly of the functional complex, but so far very little structural information has been reported for any of them. We describe the crystal structure of a large fragment of one subunit (PA) of influenza A RNA polymerase bound to a fragment of another subunit (PB1). The carboxy-terminal domain of PA forms a novel fold, and forms a deep, highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by forming a 3(10) helix.
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PMID:The structural basis for an essential subunit interaction in influenza virus RNA polymerase. 1866 Aug 1

Strong determinants of the host range of influenza A viruses have been identified on the polymerase complex formed by the PB1, PB2, and PA subunits and on the nucleoprotein (NP). In the present study, molecular mechanisms that may involve these four core proteins and contribute to the restriction of avian influenza virus multiplication in human cells have been investigated. The efficiencies with which the polymerase complexes of a human and an avian influenza virus isolate assemble and interact with the viral NP and cellular RNA polymerase II proteins were compared in mammalian and in avian infected cells. To this end, recombinant influenza viruses expressing either human or avian-derived core proteins with a PB2 protein fused to the One-Strep purification tag at the N or C terminus were generated. Copurification experiments performed on infected cell extracts indicate that the avian-derived polymerase is assembled and interacts physically with the cellular RNA polymerase II at least as efficiently as does the human-derived polymerase in human as well as in avian cells. Restricted growth of the avian isolate in human cells correlates with low levels of the core proteins in infected cell extracts and with poor association of the NP with the polymerase compared to what is observed for the human isolate. The NP-polymerase association is restored by a Glu-to-Lys substitution at residue 627 of PB2. Overall, our data point to viral and cellular factors regulating the NP-polymerase interaction as key determinants of influenza A virus host range. Recombinant viruses expressing a tagged polymerase should prove useful for further studies of the molecular interactions between viral polymerase and host factors during the infection cycle.
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PMID:Avian Influenza A virus polymerase association with nucleoprotein, but not polymerase assembly, is impaired in human cells during the course of infection. 1901 50

A total of 1991 environmental samples of fresh avian feces and urine from several aquatic bird species in a coastal area of Northeast Germany were examined for the presence of avian influenza viruses (AIV). By real-time reverse-transcriptase polymerase chain reaction, specific for an M gene of influenza A viruses, none of 659 duck samples and only 11 (0.9%) of 1,268 geese and swan samples tested positive. Two of these were identified as H5N2 viruses of low pathogenicity. Conventional cloacal and oropharyngeal swab samples (n=1,402) collected in an adjacent coastal region in Northeast Germany from comparable species of captured or hunted birds, yielded a similar detection rate (3/901; 0.4%) for AIV-specific RNA in geese and swans, but a higher rate (4/309; 1%) for ducks. No virus isolates were obtained from either set of samples. Collection of environmental avian samples was simple and cost effective and also allowed us to regulate sample sizes over time. A species assignment of these samples was possible, provided that close presampling observation of birds at the sampling sites was secured. Environmental sampling to monitor AIV in wild bird populations may be a valid alternative to the more-invasive and capture-dependent methods based on cloacal sampling.
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PMID:Active surveillance for avian influenza virus infection in wild birds by analysis of avian fecal samples from the environment. 1939 63

Reverse transcriptase PCR designed to amplify the N1 to N9 neuraminidase (NA) genes of avian influenza viruses detected 118 of the 119 NA genes tested (99.2%) in a subtype-specific manner. This technique successfully subtyped all 167 recent avian influenza viruses isolated from birds. Subtype specificity was confirmed by sequence analyses of all 285 PCR products.
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PMID:Use of reverse transcriptase PCR to subtype N1 to N9 neuraminidase genes of avian influenza viruses. 1940 72


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