Quantification of viral RNA in blood samples of H7N1 inoculated chickens from 6 to 48 hpi.

<p>Viral RNA was detected by by quantitative real time RT-PCR (RT-qPCR) in blood samples of chickens infected with the HPAI virus H7N1 in increasing levels from 18 hpi to 48 hpi. Viral RNA levels were expressed as log10 viral RNA copies/µl. Limit of detection is indicated with the dashed line. The number of positive samples from the total number of animals is indicated above each bar.</p

Immunofluorescence staining to detect IgY leakage in EB perfused and fresh frozen brain sections.

<p>Detection of EB and IgY extravasion in the telencephalic pallium (Pall) of infected chickens at 24 and 48 hpi in comparison with a control chicken at 48 hpi (figures in the top, from A1 to C3 measure 50 µm and figures in the bottom measure 25 µm). EB extravasation (red colour) in the telencephalic pallium (Pall) was only observed in brain samples of chickens evaluated at 48 hpi (C1, C4). Images at two different magnifications showing a microvessel with a fan-like area of EB leakage (C1, C4). No EB extravasation was observed in non-infected control chickens perfused with EB at 48 hpi (A1, A4), nor EB extravasation was detected on infected chickens at 24 hpi (B1, B4). Leakage of the serum protein IgY (C2, C5) was observed in the vessels and the nearest brain cells in infected chickens perfused at 48 hpi (green colour). IgY staining in control (A2, A5) and infected chickens at 24 hpi (B2, B5) was limited to the lumen of the vessels. Merged image allowed demonstrating the presence of colocalization of IgY leakage in areas of EB extravasation in chickens evaluated at 48 hpi (C3, C6). Controls chickens evaluated at 48 hpi and infected chickens at 24 hpi did not show EB leakage and the IgY staining was limited to the lumen of the vessels.</p

Distribution of the HPAI H7N1 antigen detected by immunohistochemistry in formalin fixed chicken’s brain samples.

<p>Schematic sagittal drawings of the chicken’s brain showing the distribution of the IAV antigen at 24, 36 and 48 hpi, according to the coronal levels represented in the diagrams below (A, B, C, D, E, F). In this study, the intensity of the staining was always scarce; consequently, in the diagram corresponding to 24 hpi, a dot was drawn in those regions where a positive cell was found. Microphotography 1. shows an endothelial cell (black arrow) with positive viral antigen staining in the nucleus (10 µm). At 36 and 48 hpi, the intensity of influenza virus antigen staining was slight, varying the number of cell from 2 to 80 positive cells per foci. Bilateral staining was only found in the Rot (thalamus- p2) at these hours (labelled in yellow in the diagram of the left). Microphotography 2. shows a neuron surrounded by several positive glial cells (arrowhead) beside a disrupted capillary (black arrow) at 36 hpi, located in the Rot (p2) (25 µm). Positive viral antigen was detected in few ependymal cells until 48 hpi. Microphotography 3. shows the presence of viral antigen in endothelial cells (black arrow), glial cells (arrowhead), neurons, and ependymal cells (white arrow) (25 µm).</p

Immunofluorescence staining to detect IAV antigen in brain samples of EB-perfused H7N1 infected chickens.

<p>IAV antigen (green) labelling was codetected in areas of EB extravasation (red) and extends surrounding the leakage area in a chicken at 48 hpi, 50 µm.</p

Experimental design. Procedures followed for each group of chickens included in the study.

<p>Chickens were divided in two groups; the first consisted of 46 chickens that were inoculated with 10⁶ ELD₅₀ of H7N1 A/Chicken/Italy/5093/99 HPAI virus, while the second group of 18 chickens were used as non-infected control. In turn, the inoculated chickens were divided in three groups (A, B, and C), as well the non-infected chickens that were divided en two groups. Group A consists of 18 H7N1 inoculated chickens and 6 non-infected chickens that were perfused with EB at 6, 12, 18, 24, 36 and 48 hpi and their brains were frozen. Group B includes 16 H7N1 inoculated and 12 non-infected chickens that were perfused with PBS and their brains were frozen. Group C contains 12 chickens inoculated with H7N1 and which brains were fixed in formalin.</p

Detection of the TJ protein ZO-1 and IAV antigen in samples of H7N1 infected chickens.

<p>Immunofluorescense staining of fresh frozen brain samples of infected chickens at 36 hpi showing the loss of ZO-1 marker (C) (labelled in red colour) in a focus of gliosis (B) positive for influenza viral antigen (A) (labelled in green colour) found in the telencephalic pallium (Pall) (50 µm). Merged image showing the absent of ZO-1 marker labelling that is affecting specifically the area of gliosis where IAV antigen was found (D).</p

Lethality in chickens after challenge with H5N1 HPAIV.

<p>Survival curves (in percentages) of SPF-chickens from G1 (pre-exposed to H7N2 LPAIV and subsequently infected with H7N1 HPAIV) and G3 (positive control) after H5N1 HPAIV-challenge.</p

Presence of specific antibodies against H7-, N1- and N2- evaluated by ELISA.

<p>Pooled sera from chickens were taken 15 days post-H7N2 LPAIV exposure and 10 days post-H7N1 HPAIV challenge and were tested for binding to (A) H7 hemagglutinin, (B) N1 or (C) N2 neuraminidases by ELISA.</p

Experimental design.

<p>Thirty 2-week old SPF-chickens were randomly distributed into three groups. Animals received the first inoculum (day 0) and 2 weeks later (day 15), birds were challenged with the respective inoculum 2. Six birds from G1 and G2 were consecutively infected 2 weeks later (day 30) with the third inoculum.</p><p>Abbreviations: LPAIV = low pathogenic avian influenza virus; HPAIV = highly pathogenic avian influenza virus.</p>a<p>Chickens from G1 were inoculated intranasally with LPAIV A/<i>Anas plathyrhynchos</i>/Spain/1877/2009 (H7N2) (10^5.5 ELD₅₀). Birds from G2 and G3 received saline solution.</p>b<p>Chickens from G1 and G2 were intranasally challenged with HPAIV A/FPV/Rostock/34 (H7N1) (10^5.5 ELD₅₀) 15 days after the pre-exposure to H7N2 LPAIV. Birds from G3 received saline solution.</p>c<p>Chickens from G1 and G3 were inoculated intranasally with 10^4.5 ELD₅₀ of A/Great crested grebe/Basque Country/06.03249/2006 (H5N1) 15 days after the challenge with H7N1 HPAIV.</p

Serological status, as determined by hemagglutination inhibition, of chickens 15 days after experimental pre-exposure to H7N2 LPAIV^a and 10 days after challenge with H7N1 HPAIV^b.

<p>Sera from the animals were tested against H7 and H5 antigens.</p>a<p>Chickens were inoculated intranasally with A/<i>Anas plathyrhynchos</i>/Spain/1877/2009 (H7N2) (10^5.5 ELD₅₀). Serologic data from six randomly selected birds per group are presented.</p><p>Due to the lack of seroconversion, only four animals from the naïve group are represented in the table.</p>b<p>Chickens were challenged intranasally with A/FPV/Rostock/34 (H7N1) (10^5.5 of ELD₅₀) 15 days after the pre-exposure to H7N2 LPAIV.</p>c<p>HI titers ≥8 were considered positive.</p>d<p>HI using antigen against H7N3 subtype.</p>e<p>HI using antigen against H5N1 aubtype.</p>†<p> = succumbed to H7N1 HPAIV-infection.</p