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)

Avian influenza virus (AIV) was recovered from the internal contents of eggs, including mixture of albumen and allantoic fluid, and from the oviduct of naturally infected Japanese quail (Coturnix coturnix japonica) flocks in the southern part of Thailand. The virus titers of 10(4.6)-10(6.2) ELD(50)/mL were directly measured from the internal content of infected eggs. The virus was isolated by chorioallantoic sac inoculation of embryonating chicken eggs. Infected allantoic fluid was identified as hemagglutinating virus and then was indicated the presence of H5 hemagglutinin. The virus was confirmed to be H5N1 subtype influenza A virus by reverse transcriptase-polymerase chain reaction. Additionally, real-time reverse transcriptase-polymerase chain reaction assay could specifically detect influenza virus subtype H5. Furthermore, indirect fluorescent antibody (IFA) test by using specific anti-influenza A monoclonal antibody indicated that virus antigens were detected in the parenchyma of multiple tissues. Systemic localization of viral antigen detected was certainly considered to be viremic stage. In addition, influenza virus antigen was also detected by IFA in allantoic fluid sediments isolated from internal content of egg or oviduct. The conclusion of isolated AIV type A subtype H5N1 from these two infected materials was correlated to the viremic stage of infection because the virus antigens could be observed in almost all tissues. Conclusively, the need for adequate safeguards to prevent contamination and spread of the virus to the environment during movement of eggs--including hatching eggs, cracked eggs, and other relevant infected materials-- or egg consumption from area of outbreak is emphasized and must not be ignored for the reasons of animal, public, and environmental health.
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PMID:Isolation of avian influenza virus A subtype H5N1 from internal contents (albumen and allantoic fluid) of Japanese quail (Coturnix coturnix japonica) eggs and oviduct during a natural outbreak. 1713 8

Real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) is becoming an established first-line diagnostic assay as well as a precise quantification tool for avian influenza virus detection. However, there remain some limitations. First, we show that the sensitivity of RRT-PCR influenza detection can be 10- to 100-fold inhibited in oropharyngeal and cloacal swabs. Adding 0.5 U of heat-activated Taq DNA polymerase successfully reverses PCR inhibition. Second, an excellent strategy for detecting false negative samples is the coamplification of an internal control from each sample. We developed a universal avian endogenous internal control (bird beta-actin) and apply it to influenza A diagnosis. Moreover, this internal control proves useful as a normalizer control for virus quantification, because beta-actin gene expression does not change in infected vs. uninfected ducks. A combined panel of wild bird cloacal swabs, wild bird tissue samples, experimental duck swabs, and experimental duck and chicken tissue samples was used to validate the endogenous control. The application of an endogenous internal control proves an excellent strategy both for avoiding false negative diagnostic results and for standardizing virus quantification studies.
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PMID:A universal avian endogenous real-time reverse transcriptase-polymerase chain reaction control and its application to avian influenza diagnosis and quantification. 1749 56

Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.
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PMID:Rapid detection of highly pathogenic avian influenza H5N1 virus by TaqMan reverse transcriptase-polymerase chain reaction. 1749 86

Real time reverse transcriptase (RRT)-polymerase chain reaction (PCR) for the detection of Eurasian H5 avian influenza virus (AIV) isolates was adapted from an existing protocol, optimized, and validated using a number of genetically diverse H5 isolates (n = 51). These included 34 "Asian lineage" H5N1 highly pathogenic avian influenza (HPAI) viruses (2004-2006), plus 12 other H5 isolates from poultry outbreaks and wild birds in the Eastern Hemisphere (1996-2005). All 51 were positive by H5 Eurasian RRT-PCR. Specificity was assessed by testing representative isolates from all other AL virus subtypes (n = 52), non-AI avian pathogens (n = 8), plus a negative population of clinical specimens derived from AI-uninfected wild birds and poultry (n = 604); all were negative by H5 Eurasian RRT-PCR. RNA was directly extracted from suspect HPAI H5N1 clinical specimens (Africa, Asia, and Europe; 2005-2006; n = 58) from dead poultry and wild birds, and 55 recorded as positive by H5 Eurasian RRT-PCR: Fifty-one of these 55 were in agreement with positive AIV isolation in embryonated chickens' eggs. H5 Eurasian RRT-PCR was invaluable in H5 outbreak diagnosis and management by virtue of its rapidity and high degree of sensitivity and specificity. This method provides a platform for automation that can be applied for large-scale intensive investigations, including surveillance.
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PMID:Validated H5 Eurasian real-time reverse transcriptase-polymerase chain reaction and its application in H5N1 outbreaks in 2005-2006. 1749 87

Prevalence of avian influenza infection in free-range mule ducks (a cross between Muscovy [Cairina moschata domesticus] and Pekin ducks [Anas platyrhychos domesticus]) is a matter of concern and deserves particular attention. Thus, cloacal swabs were collected blindly from 30 targeted mule flocks at 4, 8, and 12 wk of age between October 2004 and January 2005. They were stored until selection. On the basis of a positive H5 antibody detection at 12 wk of age with the use of four H5 antigens, the samples from eight flocks were selectively analyzed. Positive samples were first screened with a matrix gene-based real-time reverse transcriptase-polymerase chain reaction assay before virus isolation. Eight avian influenza subtypes (H5N1, H5N2, H5N3, H6N2, H6N8, and H11N9) and three avian paramyxovirus type 1 viruses were isolated. All 11 are characterized as low pathogenicity (LP) and avirulent, respectively, by in vivo tests and, when relevant, nucleotide sequencing of the hemagglutinin (or fusion [F]) protein cleavage site. Regarding H5 isolates, all of their eight genes belong to the avian lineage and some particular genetic traits were determined. H5 genes were fully sequenced and phylogenetically analyzed; they all belong to the Eurasian lineage, lack additional glycosylation sites, and do not cluster, suggesting separate introductions from the wild reservoir. None were grouped with the Asian isolates. The N1 gene (H5N1 isolate) was very close genetically to an Italian LP-H7N1 gene. Antigenic relationships between these H5 isolates and others were assessed comparatively by crossed hemagglutination inhibition tests. All these data are very useful to control the evolution of H5 viruses at the genetic and antigenic level to better understand the source of new outbreaks (new introductions from wild birds or the result of spread among poultry) and to assess the immunity afforded by available vaccines. These data are useful also to update antigens for avian influenza survey and to choose the most suitable vaccine in the case of preventive vaccination of ducks.
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PMID:Virologic findings in selected free-range mule duck farms at high risk for avian influenza infection. 1749 95

Following the avian influenza (AI) epidemics occurring in different areas of the world, a surveillance program funded by the Italian Ministry of Health was implemented. In the framework of this program, an investigation of wild birds was carried out to assess the circulation of AI viruses in their natural reservoir. More than 3000 samples, mainly cloacal swabs, were collected from migratory wild birds belonging to the orders Anseriformes and Charadriiformes. Samples were screened by means of a real-time reverse transcriptase polymerase chain reaction (RRT-PCR), then processed for attempted virus isolation in embryonated fowl's specific pathogen-free eggs. Approximately 5% of the samples were positive for type A influenza viruses by RRT-PCR, and from 14 of those samples AI viruses were isolated and fully characterized. The isolates, belonging to 8 different avian influenza virus subtype combinations (H10N4, H1N1, H4N6, H7N7, H7N4, H5N1, H5N2, and H5N3), were obtained from migratory Anseriformes.
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PMID:Influenza virus surveillance in wild birds in Italy: results of laboratory investigations in 2003-2005. 1749 96

Since the reemergence of highly pathogenic avian influenza virus H5N1, it caused disease in 20 people with 13 deaths in mainland of China. On February 21, 2006, the first suspected human case in Zhejiang province was reported. Pathogenic analyses, including reverse transcriptase polymerase chain reaction (RT-PCR), real-time RT-PCR, and virus isolation, were carried out to confirm the pathogen from tracheal aspirate specimen. In addition, antibody in serum sample was detected using hemagglutination-inhibition (HI). Results revealed that nucleic acid extracted from the tracheal aspirate specimen was positive for H5N1 avian influenza virus and influenza virus type A. The H5N1 virus strain named A/Zhejiang/16/06 (H5N1) was isolated. The titers of HI antibody for H5N1 avian influenza virus were 1:320 and 1:640, respectively. The sequenced genes were all avian origin. Phylogenetic analyses between the A/Zhejiang/16/06 and other H5N1 influenza viruses were also included.
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PMID:Pathogenic and molecular characterization of the H5N1 avian influenza virus isolated from the first human case in Zhejiang province, China. 1750 92

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10(+1.2) egg infectious dose (EID)(50)/0.2 ml and 10(+2.2) EID(50)/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.
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PMID:Real-time reverse transcription-polymerase chain reaction detection of Newcastle disease virus using light upon extension fluorogenic primers. 1760 51

Following the avian influenza epidemics that occurred in Italy between 1997 and 2003, the Italian Ministry of Health in collaboration with veterinary authorities promoted, funded and implemented a national surveillance programme. The main objectives of the surveillance effort were to identify avian influenza viruses circulating in wild birds and to investigate the role of backyard poultry flocks in the dynamics of infection in a densely populated poultry area. Over 2 years (2004 to 2006), 164 backyard flocks and 4083 wild birds (mainly migratory Anseriformes and Charadriiformes) were sampled in three regions in the North of Italy. Samples collected were screened by means of real-time reverse transcriptase-polymerase chain reaction and the positive samples were processed for attempted virus isolation in embryonated fowl's specific pathogen free eggs. At the end of the study period, 27 low-pathogenic avian influenza viruses had been isolated from backyard flocks and 49 strains obtained from wild birds. Of these, 26 belonged to the H5 or H7 subtype and were closely related to contemporary low-pathogenic strains of Eurasian lineage. The findings confirm that backyard free-range farming is at high risk for avian influenza virus introduction, and confirm the role of wild waterfowl in the introduction and perpetuation of low-pathogenic avian influenza viruses during the winter season in Southern Europe.
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PMID:Active surveillance for avian influenza viruses in wild birds and backyard flocks in Northern Italy during 2004 to 2006. 1762 Jan 82

The diagnosis of avian influenza (AI) virus infections, even highly pathogenic AI (HPAI), represents a considerable challenge due to the lack of pathognomonic or specific clinical signs and their variation in different avian hosts plus the marked antigenic variation amongst influenza A viruses. Conventional laboratory techniques involve the isolation, identification and characterization (including virulence estimates) of the virus. While this has proven successful in the past and remains the method of choice, for at least the initial outbreak, the delays associated with conventional diagnosis are often considered unacceptable for the application of control measures, especially stamping out policies, and there is an overwhelming demand for rapid results. More and more, molecular biological techniques are being used and in particular reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR technologies are being employed for rapid diagnosis. In this paper, clinical signs, the molecular basis for virulence of AI viruses, international definitions, conventional diagnosis and the use of molecular techniques are reviewed and discussed.
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PMID:Avian influenza - diagnosis. 1820 22


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