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    Structured Review

    Thermo Fisher h5n1 viruses
    Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with <t>H5N1</t> (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B . ( C . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p
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    1) Product Images from "Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1"

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25073-9

    Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with H5N1 (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B . ( C . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p
    Figure Legend Snippet: Influenza A virus (IAV) infection inhibits expression of DNA methyltransferase 1 (DNMT1), resulting in up-regulation of miR-203. ( A ) A549 cells were infected with H5N1 (multiplicity of infection (MOI) = 2) viruses, and abundance of mRNAs encoding three DNA methyltransferases (DNMT1, DNMT3a, and DNMT3b) was measured at different times (0, 2, 4, 6, 12, and 24 h). ( B . ( C . ( D and E ) A549 cells were transfected for 24 h with the pCMV3-DNMT1 plasmid ( D ) or si-DNMT1 ( E ). Next, cells were mock-infected or infected with H5N1 (MOI = 2) viruses for another 24 h to detect abundance of miR-203. The empty vector (pCMV3-empty) and si-control served as negative controls. Data are expressed as the mean + SD of three independent experiments. * p

    Techniques Used: Infection, Expressing, Transfection, Plasmid Preparation

    Type I interferon (IFN) participates in transcriptional regulation of miR-203 to promote its expression directly. ( A ) A549 cells were infected with H5N1 (MOI = 2) viruses and harvested at different times (2, 6, 12, and 24 h). The abundance of IFN-α and IFN-β mRNA was measured by quantitative real-time PCR (qPCR). ( B and C ) A549 cells ( B ) and Vero cells ( C ) were treated with IFN-α (2000 units/ml) for 12 h, and expression of miR-203 was measured by qPCR. (D) A549 cells were co-transfected for 24 h with luciferase reporter vectors (pGL3-promoter) and a pRL-TK plasmid (pGL3-basic vectors were used as a negative control). Then, cells were treated with IFN-α for another 12 h followed by measurement of luciferase activity in a dual-luciferase assay. The results are presented as the normalized ratio of Firefly to Renilla luciferase activity. ( E ) Vero cells were mock-infected or infected with H5N1 (MOI = 2) viruses. Cells were harvested at different times (12, 24, 36, and 48 h), and abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p
    Figure Legend Snippet: Type I interferon (IFN) participates in transcriptional regulation of miR-203 to promote its expression directly. ( A ) A549 cells were infected with H5N1 (MOI = 2) viruses and harvested at different times (2, 6, 12, and 24 h). The abundance of IFN-α and IFN-β mRNA was measured by quantitative real-time PCR (qPCR). ( B and C ) A549 cells ( B ) and Vero cells ( C ) were treated with IFN-α (2000 units/ml) for 12 h, and expression of miR-203 was measured by qPCR. (D) A549 cells were co-transfected for 24 h with luciferase reporter vectors (pGL3-promoter) and a pRL-TK plasmid (pGL3-basic vectors were used as a negative control). Then, cells were treated with IFN-α for another 12 h followed by measurement of luciferase activity in a dual-luciferase assay. The results are presented as the normalized ratio of Firefly to Renilla luciferase activity. ( E ) Vero cells were mock-infected or infected with H5N1 (MOI = 2) viruses. Cells were harvested at different times (12, 24, 36, and 48 h), and abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Negative Control, Activity Assay

    IAV-mediated demethylation of the miR-203 promoter region up-regulates expression of miR-203. ( A ) Bisulfite sequencing PCR of the miR-203 promoter region was performed in A549 cells mock-infected or infected with H5N1 (MOI = 2) viruses for 24 h. The sequenced region covered −600 to −10 bp within the miR-203 promoter region. Three lines denote three replicates in per group. Open circles, unmethylated; solid circles, methylated. ( B ) A549 cells were treated for 48 h with 5-aza-2′-deoxycytidine, a methylation inhibitor, at a final concentration of 0.5, 1, or 2 μM, and total RNA was purified to analyze abundance of miR-203 by quantitative real-time PCR (qPCR). ( C ) Vero cells were treated for 48 h with 5-aza-2′-deoxycytidine (final concentration, 1 μM) and total RNA purified. The abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p
    Figure Legend Snippet: IAV-mediated demethylation of the miR-203 promoter region up-regulates expression of miR-203. ( A ) Bisulfite sequencing PCR of the miR-203 promoter region was performed in A549 cells mock-infected or infected with H5N1 (MOI = 2) viruses for 24 h. The sequenced region covered −600 to −10 bp within the miR-203 promoter region. Three lines denote three replicates in per group. Open circles, unmethylated; solid circles, methylated. ( B ) A549 cells were treated for 48 h with 5-aza-2′-deoxycytidine, a methylation inhibitor, at a final concentration of 0.5, 1, or 2 μM, and total RNA was purified to analyze abundance of miR-203 by quantitative real-time PCR (qPCR). ( C ) Vero cells were treated for 48 h with 5-aza-2′-deoxycytidine (final concentration, 1 μM) and total RNA purified. The abundance of miR-203 was measured by qPCR. Data are expressed as the mean + SD of three independent experiments. * p

    Techniques Used: Expressing, Methylation Sequencing, Polymerase Chain Reaction, Infection, Methylation, Concentration Assay, Purification, Real-time Polymerase Chain Reaction

    Influenza A virus (IAV) infection of A549 cells up-regulates expression of miR-203. ( A ) Venn diagram illustrates the numbers of miRNAs identified in each group and the number of common miRNAs within the four groups. ( B ) Heatmap of miRNA expression in A549 cells infected with H1N1 (501) and H5N1 viruses for 24 or 48 h. Each value for the virus/mock average signal ratio was expressed as log10 when drawing the Heatmap. Red denotes up-regulation, while green denotes down-regulation. ( C ) A549 cells were mock-infected or infected with H1N1 (501), H1N1 (PR8), H3N2, H5N1, or H7N9 for 24 or 48 h, and abundance of miR-203 was assessed by quantitative real-time PCR (qPCR). ( D and E ) A549 cells were mock-infected or infected with H1N1 (501) ( D ) or H5N1 viruses ( E ). Cells were harvested at different times (12, 24, 36, and 48 h), and dynamic changes in miR-203 expression were assessed by qPCR. Data are expressed as the mean ± SD of three independent experiments. * p
    Figure Legend Snippet: Influenza A virus (IAV) infection of A549 cells up-regulates expression of miR-203. ( A ) Venn diagram illustrates the numbers of miRNAs identified in each group and the number of common miRNAs within the four groups. ( B ) Heatmap of miRNA expression in A549 cells infected with H1N1 (501) and H5N1 viruses for 24 or 48 h. Each value for the virus/mock average signal ratio was expressed as log10 when drawing the Heatmap. Red denotes up-regulation, while green denotes down-regulation. ( C ) A549 cells were mock-infected or infected with H1N1 (501), H1N1 (PR8), H3N2, H5N1, or H7N9 for 24 or 48 h, and abundance of miR-203 was assessed by quantitative real-time PCR (qPCR). ( D and E ) A549 cells were mock-infected or infected with H1N1 (501) ( D ) or H5N1 viruses ( E ). Cells were harvested at different times (12, 24, 36, and 48 h), and dynamic changes in miR-203 expression were assessed by qPCR. Data are expressed as the mean ± SD of three independent experiments. * p

    Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction

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    Article Snippet: .. Generation of a recombinant baculovirus The pFastBac™Dual-CMV/THB-P10/CTLT vector was transformed into E. coli DH10Bac/BmNPV provided by Prof. WB Wang of Jiangsu University to generate a recombinant Bacmid-CMV/THB-P10/CTLT using the Bac-To-Bac baculovirus expression system (Invitrogen, Frederick, MD, USA) following the manufacturer’s instructions. ..

    Plasmid Preparation:

    Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models
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    Transformation Assay:

    Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models
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    Western Blot:

    Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models
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    BAC Assay:

    Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models
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    Expressing:

    Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models
    Article Snippet: .. Generation of a recombinant baculovirus The pFastBac™Dual-CMV/THB-P10/CTLT vector was transformed into E. coli DH10Bac/BmNPV provided by Prof. WB Wang of Jiangsu University to generate a recombinant Bacmid-CMV/THB-P10/CTLT using the Bac-To-Bac baculovirus expression system (Invitrogen, Frederick, MD, USA) following the manufacturer’s instructions. ..

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    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1
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    SYBR Green Assay:

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    Polymerase Chain Reaction:

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    Infection:

    Article Title: Up-regulation of microRNA-203 in influenza A virus infection inhibits viral replication by targeting DR1
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    Construction of the Baculovirus transfer vector, pFastBac™DuaI-CMV/THB-P10/CTLT, and identification of the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT. A, Construction of the Baculovirus transfer vector, pFastBac™DuaI-CMV/THB-P10/CTLT, a coding sequence (CTLT) of T lymphocyte epitopes from H1HA, H9HA, and H7HA AIV subtypes was controlled by the baculovirus P10 promoter, and a coding sequence (THB) of B cell epitopes from the H1HA, H9HA, and H7HA AIV subtypes was driven by the CMV promoter. B-1, control BmN cells; B-2, the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT-infected BmN cells. C-1, the CMV promoter amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lane 1: BmNPV; and lanes 2–4: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT. C-2, the THB fragment amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lanes 1–3: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT; lane 4: pFastBacTMDuaI-CMV/THB-P10/CTLT; lane 5: BmNPV. C-3, the CTLT fragment amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lanes 1–3: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT; lane 4: pFastBacTMDuaI-CMV/THB-P10/CTLT; lane 5: BmNPV

    Journal: Virology Journal

    Article Title: AIV polyantigen epitope expressed by recombinant baculovirus induces a systemic immune response in chicken and mouse models

    doi: 10.1186/s12985-020-01388-w

    Figure Lengend Snippet: Construction of the Baculovirus transfer vector, pFastBac™DuaI-CMV/THB-P10/CTLT, and identification of the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT. A, Construction of the Baculovirus transfer vector, pFastBac™DuaI-CMV/THB-P10/CTLT, a coding sequence (CTLT) of T lymphocyte epitopes from H1HA, H9HA, and H7HA AIV subtypes was controlled by the baculovirus P10 promoter, and a coding sequence (THB) of B cell epitopes from the H1HA, H9HA, and H7HA AIV subtypes was driven by the CMV promoter. B-1, control BmN cells; B-2, the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT-infected BmN cells. C-1, the CMV promoter amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lane 1: BmNPV; and lanes 2–4: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT. C-2, the THB fragment amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lanes 1–3: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT; lane 4: pFastBacTMDuaI-CMV/THB-P10/CTLT; lane 5: BmNPV. C-3, the CTLT fragment amplified from the recombinant Baculovirus BmNPV-CMV/THB-P10/CTLT; lane M: DNA marker; lanes 1–3: P1, P2, and P3 BmNPV-CMV/THB-P10/CTLT; lane 4: pFastBacTMDuaI-CMV/THB-P10/CTLT; lane 5: BmNPV

    Article Snippet: Generation of a recombinant baculovirus The pFastBac™Dual-CMV/THB-P10/CTLT vector was transformed into E. coli DH10Bac/BmNPV provided by Prof. WB Wang of Jiangsu University to generate a recombinant Bacmid-CMV/THB-P10/CTLT using the Bac-To-Bac baculovirus expression system (Invitrogen, Frederick, MD, USA) following the manufacturer’s instructions.

    Techniques: Plasmid Preparation, Recombinant, Sequencing, Infection, Amplification, Marker

    Short-term heat shock decreased the virulence of H5N1 HPAIV in infected mice . Mice in the heat-shocked group were exposed to 39°C for 4 h before being infected with 3 LD 50 H5N1 HPAIV intranasally. Mice were sacrificed at Days 1, 3, and 6 post-infection for harvesting of lung tissues. Representative sections from each group were stained by H E (Bar = 50 μm). The open arrow indicates interstitial edema and inflammatory cell infiltration around small blood vessels; the solid arrow indicates dropout of mucous epithelium in the bronchioles; the open triangle indicates pulmonary congestion; the solid triangle indicates infiltration of the alveolar lumen by erythrocytes; the star indicates infiltration of the alveolar lumen by inflammatory cells (A) . Three mice in each group were euthanized for measurement of viral titer in the lungs by plaque assay (B) , and real-time PCR (C) (** p

    Journal: Frontiers in Microbiology

    Article Title: Short-Term Heat Shock Affects Host–Virus Interaction in Mice Infected with Highly Pathogenic Avian Influenza Virus H5N1

    doi: 10.3389/fmicb.2016.00924

    Figure Lengend Snippet: Short-term heat shock decreased the virulence of H5N1 HPAIV in infected mice . Mice in the heat-shocked group were exposed to 39°C for 4 h before being infected with 3 LD 50 H5N1 HPAIV intranasally. Mice were sacrificed at Days 1, 3, and 6 post-infection for harvesting of lung tissues. Representative sections from each group were stained by H E (Bar = 50 μm). The open arrow indicates interstitial edema and inflammatory cell infiltration around small blood vessels; the solid arrow indicates dropout of mucous epithelium in the bronchioles; the open triangle indicates pulmonary congestion; the solid triangle indicates infiltration of the alveolar lumen by erythrocytes; the star indicates infiltration of the alveolar lumen by inflammatory cells (A) . Three mice in each group were euthanized for measurement of viral titer in the lungs by plaque assay (B) , and real-time PCR (C) (** p

    Article Snippet: Expression of the hemagglutinin (HA) gene of H5N1 influenza virus was quantified using a Power SYBR® Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA, USA).

    Techniques: Infection, Mouse Assay, Staining, Plaque Assay, Real-time Polymerase Chain Reaction

    Short-term heat shock increased the survival rate and reduced weight loss in HPAIV-infected mice . Mice were randomly divided into heat-shocked and non-shocked groups. Mice in the heat-shocked group were exposed to 39°C for 1, 2, 4, 6, and 8 h before being infected with 3 LD 50 H5N1 HPAIV intranasally. Mice in the non-shocked group were maintained at room temperature (25°C). After the treatment, all the mice were maintained at room temperature. (A) The survival rate of each group was monitored for 14 days post-infection ( n = 12–14 per group total; * p

    Journal: Frontiers in Microbiology

    Article Title: Short-Term Heat Shock Affects Host–Virus Interaction in Mice Infected with Highly Pathogenic Avian Influenza Virus H5N1

    doi: 10.3389/fmicb.2016.00924

    Figure Lengend Snippet: Short-term heat shock increased the survival rate and reduced weight loss in HPAIV-infected mice . Mice were randomly divided into heat-shocked and non-shocked groups. Mice in the heat-shocked group were exposed to 39°C for 1, 2, 4, 6, and 8 h before being infected with 3 LD 50 H5N1 HPAIV intranasally. Mice in the non-shocked group were maintained at room temperature (25°C). After the treatment, all the mice were maintained at room temperature. (A) The survival rate of each group was monitored for 14 days post-infection ( n = 12–14 per group total; * p

    Article Snippet: Expression of the hemagglutinin (HA) gene of H5N1 influenza virus was quantified using a Power SYBR® Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA, USA).

    Techniques: Infection, Mouse Assay

    Effect of heat shock treatment on HSP70 and inflammatory cytokines expression in H5N1-infected mice . Mice in the heat-shocked group were exposed to 39°C for 4 h before being infected with 3 LD 50 H5N1 HPAIV intranasally. Mice in the non-shocked group were maintained at room temperature (25°C). After infection, all the mice were maintained at room temperature. Mice were sacrificed at Days 0, 1, 3, and 6 post-infection for lung tissues harvest. HSP70 expression was determined by qPCR (A) and western blot (B) . IL-6, TNF-α, IFN-β, and IFN-γ expressions were determined by qPCR (C) and ELISA (D) . Gene expression was normalized with β-actin as an internal standard ( n = 3–5, * p

    Journal: Frontiers in Microbiology

    Article Title: Short-Term Heat Shock Affects Host–Virus Interaction in Mice Infected with Highly Pathogenic Avian Influenza Virus H5N1

    doi: 10.3389/fmicb.2016.00924

    Figure Lengend Snippet: Effect of heat shock treatment on HSP70 and inflammatory cytokines expression in H5N1-infected mice . Mice in the heat-shocked group were exposed to 39°C for 4 h before being infected with 3 LD 50 H5N1 HPAIV intranasally. Mice in the non-shocked group were maintained at room temperature (25°C). After infection, all the mice were maintained at room temperature. Mice were sacrificed at Days 0, 1, 3, and 6 post-infection for lung tissues harvest. HSP70 expression was determined by qPCR (A) and western blot (B) . IL-6, TNF-α, IFN-β, and IFN-γ expressions were determined by qPCR (C) and ELISA (D) . Gene expression was normalized with β-actin as an internal standard ( n = 3–5, * p

    Article Snippet: Expression of the hemagglutinin (HA) gene of H5N1 influenza virus was quantified using a Power SYBR® Green PCR Master Mix Kit (Applied Biosystems, Foster City, CA, USA).

    Techniques: Expressing, Infection, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

    Molecular and virological detection of highly pathogenic H5N9 in four experimentally spiked slurries (A to D), from different production facilities, with or without lime treatment. Triangles, squares, and circles indicate results of M-rRT-PCR from slurry samples without treatment, with lime treatment to achieve pH 10, or with lime treatment to achieve pH 12 (NT, T10, and T12, respectively). Filled and open symbols indicate positive and negative viral isolation of HP H5N9, respectively. The first day of the kinetic study (D0) was the day of artificial contamination and treatment. The standard deviation of the M gene rRT-PCR was estimated as a C T value of 1.2 (standard C T values between 31 and 33) using a standard RNA in a series of 20 repeated assays. Ct, cycle threshold; Undet, undetermined.

    Journal: Applied and Environmental Microbiology

    Article Title: Natural and Experimental Persistence of Highly Pathogenic H5 Influenza Viruses in Slurry of Domestic Ducks, with or without Lime Treatment

    doi: 10.1128/AEM.02288-20

    Figure Lengend Snippet: Molecular and virological detection of highly pathogenic H5N9 in four experimentally spiked slurries (A to D), from different production facilities, with or without lime treatment. Triangles, squares, and circles indicate results of M-rRT-PCR from slurry samples without treatment, with lime treatment to achieve pH 10, or with lime treatment to achieve pH 12 (NT, T10, and T12, respectively). Filled and open symbols indicate positive and negative viral isolation of HP H5N9, respectively. The first day of the kinetic study (D0) was the day of artificial contamination and treatment. The standard deviation of the M gene rRT-PCR was estimated as a C T value of 1.2 (standard C T values between 31 and 33) using a standard RNA in a series of 20 repeated assays. Ct, cycle threshold; Undet, undetermined.

    Article Snippet: M-rRT-PCR was carried out using a commercial kit (TaqMan one-step rRT-PCR master mix; Applied Biosystems) and the Applied Biosystems (ABI) 7500 Fast real-time PCR system.

    Techniques: Quantitative RT-PCR, Isolation, Standard Deviation

    Molecular and virological detection of highly pathogenic H5N8 in naturally contaminated slurries. Diamonds and triangles indicate results of weekly subsampled slurries obtained with M- and H5-rRT-PCRs, respectively. Filled and open signs indicate positive and negative viral isolation of H5N8 HP, respectively. The first day of the kinetic study (D0) was estimated as the last day when fresh fecal material from infected ducks could have been delivered into the slurry tank at the farm level. The standard deviation of the M gene rRT-PCR was estimated to be a C T value of 1.2 (standard C T values between 31 and 33) and that for the H5 gene rRT-PCR was a C T value of 1.3 (standard C T values between 34 and 36) using a standard RNA in a series of 20 repeated assays. Ct, cycle threshold; Undet, undetermined.

    Journal: Applied and Environmental Microbiology

    Article Title: Natural and Experimental Persistence of Highly Pathogenic H5 Influenza Viruses in Slurry of Domestic Ducks, with or without Lime Treatment

    doi: 10.1128/AEM.02288-20

    Figure Lengend Snippet: Molecular and virological detection of highly pathogenic H5N8 in naturally contaminated slurries. Diamonds and triangles indicate results of weekly subsampled slurries obtained with M- and H5-rRT-PCRs, respectively. Filled and open signs indicate positive and negative viral isolation of H5N8 HP, respectively. The first day of the kinetic study (D0) was estimated as the last day when fresh fecal material from infected ducks could have been delivered into the slurry tank at the farm level. The standard deviation of the M gene rRT-PCR was estimated to be a C T value of 1.2 (standard C T values between 31 and 33) and that for the H5 gene rRT-PCR was a C T value of 1.3 (standard C T values between 34 and 36) using a standard RNA in a series of 20 repeated assays. Ct, cycle threshold; Undet, undetermined.

    Article Snippet: M-rRT-PCR was carried out using a commercial kit (TaqMan one-step rRT-PCR master mix; Applied Biosystems) and the Applied Biosystems (ABI) 7500 Fast real-time PCR system.

    Techniques: Isolation, Infection, Standard Deviation, Quantitative RT-PCR

    Detection of H5N1 avian influenza A virus by one-step RT-PCR . A. Amplification of serially diluted in vitro-transcribed single-stranded RNA (lanes 2 to 11) measured by RiboGreen RNA quantitation reagent and H5N1 RNA extracted from allantoic fluid of infected egg (lane 19). The non-template control is (sterile water) illustrated as

    Journal: BMC Infectious Diseases

    Article Title: Specific detection of H5N1 avian influenza A virus in field specimens by a one-step RT-PCR assay

    doi: 10.1186/1471-2334-6-40

    Figure Lengend Snippet: Detection of H5N1 avian influenza A virus by one-step RT-PCR . A. Amplification of serially diluted in vitro-transcribed single-stranded RNA (lanes 2 to 11) measured by RiboGreen RNA quantitation reagent and H5N1 RNA extracted from allantoic fluid of infected egg (lane 19). The non-template control is (sterile water) illustrated as "NTC". The viral load is indicated by the number of copies per reaction, (lane 2) 1 × 10 9 copies per reaction, (lane 3) 1 × 10 8 copies per reaction, (lane 4) 1 × 10 7 copies per reaction, (lane 5) 1 × 10 6 copies per reaction, (lane 6) 1 × 10 5 copies per reaction, (lane 7) 1 × 10 4 copies per reaction, (lane 8) 1 × 10 3 copies per reaction, (lane 9) 1 × 10 2 copies per reaction, (lane 10) 10 copies per reaction, and (lane11) 1 copy per reaction. Viral RNA extracted from human specimens that were previously confirmed respiratory syncytial virus (RSV) B (lane 12), dengue virus 1 (lane 13), dengue virus 2 (lane 14), dengue virus 3 (lane 15), dengue virus 4 (lane 16) and severe acute respiratory syndrome (SARS) (lane 17) were also tested as known negatives. RNA extracted from healthy individual (lane 18) was also performed. B. Specific detection of H5N1 avian influenza A virus. Reference strains of different subtypes of avian influenza A viruses (lanes 20 to 24), including non-influenza viruses such as Newcastle disease virus (NDV, lane 25) are indicated. The H5N1, H3N8, H9N2 and NDV isolates were isolated from field samples by the Veterinary Research Institute, Malaysia, the H5N3 and H7N5 isolates were provided by the Department of Veterinary Pathology of Tottori University, Japan. Negative signals from non-H5N1 isolates and the non-template control (water) are shown.

    Article Snippet: The concentration of purified transcribed RNA was measured by RiboGreen RNA quantitation reagent (Invitrogen, USA) and serial dilutions of in vitro -transcribed RNA were prepared in duplicate.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, In Vitro, Quantitation Assay, Infection, Isolation