Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A) Schematic representation of the modified HA proteins of H7N7 LPAIVs and HPAIVs that were used in this study. The FLAGtag and HAtag are located downstream of the signal peptide (SP) and are connected to the mature HA1 via a linker (L) sequence. The viruses differ only at the N-terminus and at the cleavage site at the border of HA1 and HA2. (B) Chicken fibroblasts (DF-1 cells) were inoculated with H7N7-LP FLAGtag (in blue), H7N7-HP HAtag (in red), or mock-inoculated (in black) at an MOI of 0.1. Expression of the influenza virus nucleoprotein (NP), FLAGtag, and HAtag was assessed at 24 hpi by flow cytometry. (C, D) Replication kinetics in primary chicken and duck embryonic fibroblasts (CEF; DEF) following inoculation with H7N7-LP, H7N7-LP FLAGtag , H7N7-HP, or H7N7-HP HAtag at an MOI of 0.001. Infectious titers in supernatants were determined by endpoint titration in MDCK cells and expressed as log 10 TCID 50 /mL. Data are presented as arithmetic mean ± SD of log 10 transformed values from two independent experiments. The dotted lines indicate the limit of detection of the endpoint titration assay.

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Modification, Sequencing, Expressing, Virus, Flow Cytometry, Titration, Transformation Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A-D) Viral RNA quantification in oropharyngeal (OP) and cloacal (CL) swabs of chickens and ducks mono-inoculated with ~10 4 EID 50 of H7N7-HP HAtag or ~10 6 EID 50 of H7N7-LP FLAGtag . Viral RNA levels were determined by RT-qPCR targeting the influenza virus matrix segment and expressed as 40-cycle threshold (Ct). Data are depicted as arithmetic mean ± SD calculated from the number of animals indicated above the graphs (#: the number of alive animals/the total number of animals in the experiment). (E-H) Infectious virus titers in swabs from panels (A-D). Infectious titers were determined by endpoint titration in MDCK cells and expressed as log 10 TCID 50 /mL. Data are depicted as arithmetic mean ± SD calculated from log 10 transformed values from the number of animals indicated above panels (A-D). The horizontal dotted lines indicate the limit of detection of the endpoint titration assay.

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Quantitative RT-PCR, Virus, Titration, Transformation Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A, B) Viral RNA quantification in tissues harvested at 3 dpi from chickens (A) and ducks (B) inoculated with ~10 4 EID 50 of H7N7-HP HAtag or ~10 6 EID 50 of H7N7-LP FLAGtag . Viral RNA amounts were determined by RT-qPCR targeting the influenza virus matrix segment and expressed as 40-cycle threshold (Ct) normalized/gram tissue. Bars represent the arithmetic mean. The dotted lines distinguish tissues from the respiratory, digestive, and miscellaneous systems. (C, D) Infectious virus titers in tissues from panels (A, B). Infectious titers were determined by endpoint titration in MDCK cells and expressed as log 10 TCID 50 /gram tissue. Bars represent the arithmetic mean of log 10 transformed values. Dotted lines similar to (A, B). The horizontal solid lines indicate the limit of detection of the endpoint titration assay per tissue.

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Quantitative RT-PCR, Virus, Titration, Transformation Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A) Method for distinguishing H7N7-HP HAtag and H7N7-LP FLAGtag plaques based on epitope tag staining. The number of plaque forming units (PFU) per virus per sample was determined by co-staining plaques with antibodies targeting influenza virus nucleoprotein (NP) and HAtag. Plaques formed by H7N7-LP FLAGtag stain positive for NP (green). Plaques formed by H7N7-HP HAtag stain positive for both NP (green) and HAtag (red). (B) Severity of disease in six chickens co-inoculated with H7N7-HP HAtag and H7N7-LP FLAGtag . Disease severity scores (0–5) were determined thrice daily, each box representing one time point. Dotted lines indicate planned time points for euthanasia and dissection. (C) H7N7-HP HAtag and H7N7-LP FLAGtag RNA quantification in oropharyngeal (OP) and cloacal (CL) swabs from co-inoculated chickens. Viral RNA amounts were determined by HPAIV/LPAIV differentiating RT-qPCR targeting the HA cleavage site and expressed as 40-cycle threshold (Ct). (D) Infectious virus titers in the swabs from panel (C). Oropharyngeal and CL swab titers are depicted as PFU/mL as measured by the HPAIV/LPAIV differentiating plaque assay. (E) H7N7-HP HAtag and H7N7-LP FLAGtag RNA quantification in tissues harvested at 3 dpi as determined by HPAIV/LPAIV differentiating RT-qPCR targeting the HA cleavage site and expressed as 40-Ct normalized/gram tissue. The dotted lines distinguish tissues from the respiratory, digestive, and miscellaneous systems. (F) Infectious virus titers of tissues from panel (E), depicted as PFU/gram tissue as determined by the HPAIV/LPAIV differentiating plaque assay. Dotted lines are similar to (E).

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Staining, Virus, Dissection, Quantitative RT-PCR, Plaque Assay

Journal: PLOS Pathogens

Article Title: Species-specific emergence of H7 highly pathogenic avian influenza virus is driven by intrahost selection differences between chickens and ducks

doi: 10.1371/journal.ppat.1011942

Figure Lengend Snippet: (A) Degree of viral antigen expression in tissues of chickens and ducks mono-inoculated with ~10 4 EID 50 H7N7-HP HAtag or ~10 6 EID 50 H7N7-LP FLAGtag and euthanized at 3 dpi. Viral antigen expression was determined by immunohistochemical detection of the influenza virus nucleoprotein. Tissues showing no positivity were scored as (0), sporadic single positive cells as (1), multiple sites of positive cells as (2), and diffuse positivity as (3). N.P. = not performed. (B) Degree of viral antigen expression in tissues from H7N7-LP FLAGtag /H7N7-HP HAtag co-inoculated chickens and ducks euthanized at 3 dpi. Staining and scoring were performed as in (A).

Article Snippet: The plates were washed with PBS, and stained overnight at 4°C with a mixture of primary antibodies diluted in PBS with 0.1% bovine serum albumin (BSA): 1 μg/mL anti-influenza virus NP antibody (mouse IgG2α; ATCC HB-65) and 44 ng/mL anti-HAtag antibody (rabbit IgG; C29F4; Cell Signaling).

Techniques: Expressing, Immunohistochemical staining, Virus, Staining

Journal: Journal of Biological Chemistry

Article Title: Positive and Negative Modulation of the Transcriptional Activity of the ETS Factor ESE-1 through Interaction with p300, CREB-binding Protein, and Ku 70/86

doi: 10.1074/jbc.m401356200

Figure Lengend Snippet: FIG. 3. Co-immunoprecipitation of ESE-1 and p300 and CBP. Co-immunoprecipitation was performed using 293T cells expressing p300 or HA-CBP plus pCDNA3.1–5FLAG empty or FLAG-ESE-1. The expressions of FLAG-ESE-1, p300, and HA-CBP were confirmed in the total lysate (i.e. the input used for the immunoprecipitation) by Western blot using anti-FLAG antibody M2, anti-p300 antibody N-15, and an- ti-HA antibody, respectively (A). FLAG-ESE-1 was immunoprecipitated using anti-FLAG antibody M2-conjugated agarose beads. Co-immuno- precipitated products were separated by SDS-PAGE on a 6% polyacryl- amide gel for the detection of p300 and HA-CBP using N-15 and an- ti-HA antibodies and on a 10% polyacrylamide gel for detection of FLAG-ESE-1 using antibody M2 (B).

Article Snippet: Ku86 was also analyzed using mouse anti-Ku86 antibody (BD Biosciences/Pharmingen). p300 and HAtagged CBP were analyzed using rabbit polyclonal anti-p300 antibody N15 and mouse anti-HA antibody or rabbit polyclonal anti-CBP C-20 (Santa Cruz Biotechnology), respectively.

Techniques: Immunoprecipitation, Expressing, Western Blot, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Positive and Negative Modulation of the Transcriptional Activity of the ETS Factor ESE-1 through Interaction with p300, CREB-binding Protein, and Ku 70/86

doi: 10.1074/jbc.m401356200

Figure Lengend Snippet: FIG. 4. p300 and CBP synergize with ESE-1 to transactivate the PSP promoter. The effects of p300 and CBP on the transcription activity of ESE-1 were studied using the luciferase reporter assay. Expression vectors encoding p300 and CBP were co-transfected into 293T cells with empty pCDNA3.1–5FLAG vector or FLAG-ESE-1 plus the PSP-luciferase reporter (A). Dosages of p300CBP were used at ratios of p300CBP:ESE-1 of 1:1, 3:1, and 10:1. The amount of DNA in the figure indicates the amount used for a single well on a 24-well plate. At 48 h after the transfection, the cells were collected for the luciferase assay, and the value was normalized by the internal control (CMV--galactosidase). The error bars represent S.D. (n 3) (A). To determine whether endogenous p300CBP influences ESE-1 activity, E1A was expressed in 293T cells to sequester p300CBP in the reporter assays (B). To further study the potential effects of interacting proteins on the ESE-1 activity, we determined the expressions of ESE-1 and its interacting proteins, p300, CBP, Ku70, and Ku86, in HUVEC challenged with inflammatory cytokine IL-1. The IL-1 induced ESE-1 expression was confirmed by reverse transcriptase (RT) PCR, whereas glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control (C). The endogenous p300 and CBP protein levels were determined by Western blot analysis using anti-p300 (N15) and anti-CBP (C-20) antibodies. -Actin was probed by anti--actin antibody as the control to ensure the equal loading. The observed increase in p300 protein level was also analyzed using ImagePro-Plus analysis and normalized by -actin signal intensities of the corresponding lanes (D).

Article Snippet: Ku86 was also analyzed using mouse anti-Ku86 antibody (BD Biosciences/Pharmingen). p300 and HAtagged CBP were analyzed using rabbit polyclonal anti-p300 antibody N15 and mouse anti-HA antibody or rabbit polyclonal anti-CBP C-20 (Santa Cruz Biotechnology), respectively.

Techniques: Activity Assay, Luciferase, Reporter Assay, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Western Blot