Influenza viral genome copies in oral and cloacal swabs following H1N1 and H1N2 infection.

<p>Oral and cloacal swabs were collected prior to and following H1N1 (A) and H1N2 (B) infection. Viral RNA genome copies were determined by quantitative real time RT-PCR assay. Error bars represent the standard deviation of mean of 18 chickens for H1N1 and 20 chickens for H1N2.</p

Heterologous cell mediated immune response following H1N1 infection.

<p>PBMCs were obtained following infection with H1N1 and then re-stimulated with H5N1 antigen. Cell proliferation (A) and IFN-γ secretion (B) were measured. The bar represents the mean with standard deviation of 4 H1N1-infected and 2 control chickens.</p

Seroconversion following H5N1 challenge.

<p>Sera from H1N1 (n = 17) or H1N2 (n = 17) pre-infected chickens and naïve contact controls (n = 4) that survived H5N1 challenge were evaluated for H5 specific antibodies by haemagglutination inhibition (HI) and virus neutralization (VN) assays. Histograms represent means of HI titres (black histograms) and VN titres (open histograms) and bars represent standard error of means.</p

Antibodies from H1N1 and H1N2 infected chickens reduce H5N1 plaque size.

<p>MDCK cells were infected with H5N1 in the presence of sera from H1N1-infected (open histograms), H1N2-infected (grey histograms) or naïve (black histograms) chicken and plaque size (mm) determined after 3 days. Data represents mean and error bars are standard deviation for sera from at least 5 chickens per group.</p

IFN-α2a does not clear KFDV infection in BHK-21 cells.

<p>BHK-21 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging Cell monolayers were photographed prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p

Highly Pathogenic H5N1 and Novel H7N9 Influenza A Viruses Induce More Profound Proteomic Host Responses than Seasonal and Pandemic H1N1 Strains

Influenza A viruses (IAV) are important human and animal pathogens with potential for causing pandemics. IAVs exhibit a wide spectrum of clinical illness in humans, from relatively mild infections by seasonal strains to acute respiratory distress syndrome during infections with some highly pathogenic avian influenza (HPAI) viruses. In the present study, we infected A549 human cells with seasonal H1N1 (sH1N1), 2009 pandemic H1N1 (pdmH1N1), or novel H7N9 and HPAI H5N1 strains. We used multiplexed isobaric tags for relative and absolute quantification to measure proteomic host responses to these different strains at 1, 3, and 6 h post-infection. Our analyses revealed that both H7N9 and H5N1 strains induced more profound changes to the A549 global proteome compared to those with low-pathogenicity H1N1 virus infection, which correlates with the higher pathogenicity these strains exhibit at the organismal level. Bioinformatics analysis revealed important modulation of the nuclear factor erythroid 2-related factor 2 (NRF2) oxidative stress response in infection. Cellular fractionation and Western blotting suggested that the phosphorylated form of NRF2 is not imported to the nucleus in H5N1 and H7N9 virus infections. Fibronectin was also strongly inhibited in infection with H5N1 and H7N9 strains. This is the first known comparative proteomic study of the host response to H7N9, H5N1, and H1N1 viruses and the first time NRF2 is shown to be implicated in infection with highly pathogenic strains of influenza

Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.

<p>Antiviral activity of interferon-α/β against KFDV and VSV-GFP virion production.</p

Effect of Murine Type I IFNs on Recombinant Virus Replication in Mouse Macrophages

<p>RAW 264.7 cells (mouse peritoneal macrophage-derived cell line) were infected with a multiplicity of infection of 0.05. Cells were untreated (A), treated with murine IFN-α/β (500 units/ml) 2 h postinfection (B), or treated with murine IFN-α/β (500 units/ml) 12 h prior to and again 2 h after virus adsorption (C). Supernatants were collected on days 0, 1, 2, 3, and 4 postinfection and titrated by use of a focus-forming unit assay in Vero E6 cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020073#ppat-0020073-b036" target="_blank">36</a>].</p

IFN-α2a does not clear KFDV infection in A549 cells.

<p>A549 cells were infected with a 11 TCID₅₀ units (MOI of 0.00001) of the indicated virus, and either treated or mock-treated with 2, 000 U/mL of IFN-α2a (designated as P0). Monolayers were passaged when untreated controls reached CPE of 90%, 96 and 48 hpi for KFDV and VSV-GFP, respectively and 2, 000 U/mL of IFN-α2a was either added or omitted (P1) and after 72 hpi for KFDV and 48 hpi for VSV-GFP. This procedure was repeated again for passage 2 (P2). (A) Before each passage, supernatants were harvested for titration by TCID₅₀ assay determination on BHK-21 cells. The averages and standard deviation from three biological replicates are shown graphically and expressed in log₁₀ scale TCID₅₀/mL. Statistical significance is denoted as * P < 0.1, ** P < 0.05, *** P < 0.01. (B) Cell monolayers were visualized with light microscopy prior to passaging. Mock, non-IFN treated/infected controls. UI, Un-infected controls.</p

KFDV NS5 impedes the cellular antiviral state.

<p><b>(A)</b> VeroE6 (ATCC) cells were transfected with plasmid encoding KFDV NS proteins and Ebola virus VP24 and treated with 1, 000 U/mL of Universal IFN, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, pictures were taken with fluorescent (top panel) and light (bottom panel) microscopy 24 hours later. <b>(B)</b> VeroE6 (ATCC) cells were transfected with KFDV NS5-pCAGGS and treated with 1, 000 U/mL of commercially available type I IFNs, 24 hpi. After a 24-hour incubation period, cells were infected with VSV-GFP (MOI of 2) and, 24 hours later, the virus-containing supernatants were harvested for virus quantification. Dark grey bars indicate experiments in which cells were un-transfected. Light grey bars indicate NS5-expressing cells. Mock, no IFN treatment of cells lacking NS5 expression (dark gray bar) and with NS5 expression (light gray bar); UI represents uninfected/un-treated cells. Universal IFN controls included VP24-pCAGGS as anti-IFN control. The graph represents the log₁₀ scale TCID₅₀/mL averages and standard deviations from three biological repetitions. ***, Significant difference of NS5-expressing cells compared to VP24-expressing cells (P < 0.01).</p