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 objective of this research was to optimize sampling parameters for increased recovery and detection of airborne porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV). Collection media containing antifoams, activated carbons, protectants, and ethylene glycol were evaluated for direct effects on factors impacting the detection of PRRSV and SIV, including virus infectivity, viability of continuous cell lines used for the isolation of these viruses, and performance of reverse transcriptase PCR assays. The results showed that specific compounds influenced the likelihood of detecting PRRSV and SIV in collection medium. A subsequent study evaluated the effects of collection medium, impinger model, and sampling time on the recovery of aerosolized PRRSV using a method for making direct comparisons of up to six treatments simultaneously. The results demonstrated that various components in air-sampling systems, including collection medium, impinger model, and sampling time, independently influenced the recovery and detection of PRRSV and/or SIV. Interestingly, it was demonstrated that a 20% solution of ethylene glycol collected the greatest quantity of aerosolized PRRSV, which suggests the possibility of sampling at temperatures below freezing. Based on the results of these experiments, it is recommended that air-sampling systems be optimized for the target pathogen(s) and that recovery/detection results should be interpreted in the context of the actual performance of the system.
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PMID:Optimization of a sampling system for recovery and detection of airborne porcine reproductive and respiratory syndrome virus and swine influenza virus. 1682 Apr 75

We studied the occurrence of swine influenza virus (SIV) infection in piglets with respiratory symptoms resembling porcine respiratory disease complex (PRDC). A total of 106 samples including nasal swab and lung suspension from sick piglets were collected from 30 farms of medium size in the central and eastern parts of Thailand from August 2006 to February 2007. Samples were inoculated onto Mardin-Darby Canine Kidney (MDCK) cells and SIV infection was confirmed by immunofluorescent assay (IFA) and reverse transcriptase polymerase chain reaction (RT-PCR) specific for M gene. Of 106 samples, 3 pigs from 3 different farms were found to be SIV positive on all assays. The positive samples were further identified by RT-PCR as H3N2 subtype using specific primers for hemagglutinin (HA) and neuraminidase (NA) genes. SIV infection was found in 2.8% of swine suffering from respiratory distress suggesting SIV may not be the major pathogen for PRDC in the central and eastern Thailand. SIV was present in 3 of 30 farms (10%) indicating the prevalence of SIV in these regions is considerable. Since pigs are vulnerable to infection from both human and avian influenza viruses and interspecies transmission between humans and swine occurs sporadically, it is essential to continue surveillance and monitoring of SIV infection in the swine population.
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PMID:Occurrence of swine influenza virus infection in swine with porcine respiratory disease complex. 1906 93

Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.
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PMID:[Cloning and characterization of M1 gene of H3N2 subtype swine influenza virus]. 1967 Jun 34

A pandemic caused by a novel influenza A virus (H1N1) poses a serious public health threat. In this study, a real-time reverse transcriptase PCR (RT-PCR) assay based on the hemagglutinin gene was developed that discriminates the novel H1N1 from swine influenza virus, seasonal H1N1/H3N2 virus and the highly pathogenic H5N1 avian influenza virus. The sensitivity of this assay was 0.2 50% tissue culture infective dose of virus and 200 copies of in vitro-transcribed target RNA. Three hundred and forty-eight clinical specimens from suspected H1N1 patients were tested using this assay, and forty-two (12.07%) were found to be positive. Tests using the real-time PCR assay recommended by WHO and virus isolation gave identical results. This sensitive and specific real-time RT-PCR assay will contribute to the early diagnosis and control of the emerging H1N1 influenza pandemic.
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PMID:Development of a real-time RT-PCR assay for a novel influenza A (H1N1) virus. 1981 30

In the spring of 2009, a novel (H1N1) influenza A virus began to spread among humans worldwide. Although the 2009 H1N1 is related genetically to swine influenza viruses, human infection has not been connected to pig exposure. Because the virus is now circulating widely in the human population, swine herds are at increased risk of becoming infected. In order to investigate potential outbreaks of the 2009 pandemic virus in pigs, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for the detection of the (H1N1) 2009 RNA in clinical specimens was developed. To evaluate the applicability of the test as a diagnostic tool in the screening of field specimens from swine, 64 field isolates of North American swine, 5 equine and 48 avian influenza viruses collected during diagnostic investigations were analyzed retrospectively as well as samples collected during an experimental in vivo infection with two novel H1N1 isolates, A/California/04/2009 (H1N1)v virus and A/Mexico/4108/2009 (H1N1)v. The sensitivity of the qRT-PCR was shown to be higher with respect to standard techniques such as virus isolation and the reproducibility was satisfactory. The present unique and highly sensitive assay is able to detect as little as 1 x 10(1) copies of RNA per microl of template and it represents a rapid and useful approach for the screening and quantitation of (H1N1) 2009 RNA in porcine specimens.
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PMID:One-step real-time RT-PCR for pandemic influenza A virus (H1N1) 2009 matrix gene detection in swine samples. 2000 4

Nasopharyngeal swabs (n = 601) from 278 adult and 323 pediatric patients were tested within 24 h of receipt by cytospin-enhanced direct immunofluorescence antibody testing (DFA) and real-time reverse transcriptase PCR (RT-PCR) using the CDC assay. Cytospin-enhanced DFA detected 230 (84.6%) of 272 swine influenza A PCR-positive results overall but 25 (92.6%) of 27 positive results in patients less than 5 years old and 208 (96.7%) of 215 positive samples with cycle threshold values of <26.
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PMID:Cytospin-enhanced immunofluorescence and impact of sample quality on detection of novel swine origin (H1N1) influenza virus. 2004 32

M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication. It is also a target molecule of anti-virus drugs. We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+) (designated pET-28a(+)-M2) and a eukaryotic expression vector p3xFLAG-CMV-7.1 (designated p3xFLAG-CMV-7.1-M2), respectively. The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis. We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21 (DE3) strain and expressed it by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). The recombinant M2 protein was purified from the induced bacterial cells using Ni(2+) affinity chromatography. Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it. Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells. We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence. The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells. Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein, which was detectable 12 h post-infection, later than NS1, NP and M1 proteins. It would be a potential molecular indicator of late phases replication of virus. Our results would be useful for studying the biological function of M2 protein in SIV replication.
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PMID:[Characterization of M2 gene of H3N2 subtype swine influenza virus]. 2035 87

Classical swine H1N1, emerging European avian-like H1N1 and human-like H3N2 lineages are co-circulating in the swine population in China. The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an effective method for use in influenza surveillance. In this study, a multiplex RT-PCR method was developed for simultaneous identification of hemagglutinin (HA) genes derived from the three lineages of swine influenza viruses. Three primer sets were designed and aimed specifically at HA genes of these viral lineages. The specificity of the assay showed that the established methods could efficiently differentiate the HA genes of classical swine H1N1, European avian-like H1N1, and human-like H3N2 viruses while other viruses such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, and porcine circovirus type 2, could not be detected. The assay showed a sensitivity of 1 x 10(2.5) 50% egg infectious dose for each virus lineage. The comparison of the results with those obtained from the analysis of 300 swine tracheal swab samples by means of virus isolation showed a high level of agreement. This multiplex RT-PCR method provides a rapid and specific swine influenza diagnostic tool that also has the potential for investigating the epidemiology of different lineages of swine influenza virus prevalent currently in China.
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PMID:Establishment of a multiplex RT-PCR assay to detect different lineages of swine H1 and H3 influenza A viruses. 2070 Jul 59

Pandemic (H1N1) 2009 influenza has spread throughout the world since April 2009 and has caused many human deaths since its first report in humans. Pandemic (H1N1) 2009 influenza virus was first identified in a Canadian pig herd in April 2009 and has been reported in more than ten countries, including Korea. We developed a one-step multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay based on the matrix gene that discriminates pandemic (H1N1) 2009 influenza virus from endemic swine influenza viruses. The sensitivity of this assay was 100 copies of in vitro-transcribed target RNA and 0.01 tissue culture infective dose (TCID(50)/ml) of virus and was as high as those of conventional influenza A virus common matrix reverse transcriptase PCR assays and real-time reverse transcriptase PCR assays (1 to 200 copies) developed for detecting pandemic (H1N1) 2009 influenza viruses from human and pig samples. This one-step multiplex RT-PCR assay would be a good tool in monitoring pandemic (H1N1) 2009 influenza virus among pig herds on a regular basis.
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PMID:One-step multiplex reverse-transcriptase PCR for detecting pandemic (H1N1) 2009 influenza virus. 2079 77

Swine influenza viruses (SIV) have been shown to sporadically infect humans and are infrequently identified by the Influenza Division of the Centers for Disease Control and Prevention (CDC) after being received as unsubtypeable influenza A virus samples. Real-time reverse transcriptase PCR (rRT-PCR) procedures for detection and characterization of North American lineage (N. Am) SIV were developed and implemented at CDC for rapid identification of specimens from cases of suspected infections with SIV. These procedures were utilized in April 2009 for detection of human cases of 2009 A (H1N1) pandemic (pdm) influenza virus infection. Based on genetic sequence data derived from the first two viruses investigated, the previously developed rRT-PCR procedures were optimized to create the CDC rRT-PCR Swine Flu Panel for detection of the 2009 A (H1N1) pdm influenza virus. The analytical sensitivity of the CDC rRT-PCR Swine Flu Panel was shown to be 5 copies of RNA per reaction and 10(-1.3 - -0.7) 50% infectious doses (ID(50)) per reaction for cultured viruses. Cross-reactivity was not observed when testing human clinical specimens or cultured viruses that were positive for human seasonal A (H1N1, H3N2) and B influenza viruses. The CDC rRT-PCR Swine Flu Panel was distributed to public health laboratories in the United States and internationally from April 2009 until June 2010. The CDC rRT-PCR Swine Flu Panel served as an effective tool for timely and specific detection of 2009 A (H1N1) pdm influenza viruses and facilitated subsequent public health response implementation.
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PMID:Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus. 2159 60


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