Experimental conditions used for the study in simple (A) and complex (B) biotopes.

a<p>Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. Human origin strain stands for the A/Cambodia/408008/2005 strain.</p>b<p>T° = Temperature (°C).</p

Survival of infectious particles and persistence of virus RNA in complex biotopes (experiments B).

<p>B.1: complex biotopes inoculated with virus of avian or human origins at various final concentrations and maintained at 25°C. B.2: complex biotopes inoculated with virus of avian or human origins at various final concentrations and maintained at various temperatures. A: Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. H: Human origin strain stands for the A/Cambodia/408008/2005 strain.*last day of the corresponding experiment at which samples could be collected and tested.</p

Design of experiments D and laboratory results to assess the role of <i>Betta splendens</i> fish in the transmission cycle of H5N1 virus in water.

<p> The 3 horizontal rectangles represent different types of water in which animals were immersed: contaminated vs non-contaminated. Tadpoles (T) were immersed along with the male fighting fish (F) in contaminated water. Black-filled lines represent the male fighting fish and the tadpoles immersed in contaminated water from day 0 (D0). At day 5, male contaminated fish were placed over-night in clean water before being exposed to non-contaminated females in clean water. White-filled lines represent naïve female fighting fish immersed in non-contaminated water from day 0. Stars indicate collection of fish and tadpoles (when present) for testing. The two experiments are discernable and identified as D.1 and D.2. White stars correspond to detection of infectious virus in fish. Black stars indicate that H5N1 virus was not recovered from fish's organs after inoculation into embryonated eggs. Numbers included in black boxes correspond to average viral loads measured in fish (F) and tadpoles (T) that were immersed in contaminated water. Numbers in white boxes correspond to average viral loads measured in female fighting fish that were immersed in clean water and exposed to contaminated male fish. Water samples were collected from contaminated water (experiment D.1) from day 0 (D0) up to day 20 (D20). Water samples were collected from non-contaminated water (experiment D.2) from day 0 (D0) to day 12 (D12). The values presented in italic correspond to the viral load measured in water samples. When the numbers in italic are displayed in white color, this indicates that infectious virus was also detected. When the numbers in italic are written in black color, this means that the virus was not recovered after inoculation into eggs.</p

Survival of infectious particles and persistence of virus RNA in water and fauna in experiments C and D.

<p> C: water inoculated with the virus of human origin, at a final estimated concentration of 5×10⁴ EID50/mL, maintained at 25°C and containing mussels. D: water inoculated with the virus of avian origin, at a final estimated concentration of 2×10² EID50/mL, maintained at 17°C and containing fighting fish and tadpoles.*last day of the corresponding experiment at which samples could be collected and tested.</p

Survival of infectious particles and persistence of virus RNA in simple biotopes (experiments A).

<p> A.1: only water of various origins maintained at 25°C and inoculated to a final concentration of 5×10⁴ EID50/mL with H5N1 virus of animal or avian origin. A.2:water and mud containing an estimated final concentration of virus of avian origin of 5×10⁴ EID50/mL water maintained at 25°C (A.2.1) or various concentrations of viruses of avian and human origins and maintained at various temperatures (A.2.2). A: Avian origin strain stands for the A/Chicken/Cambodia/LC1AL/2007 strain. H: Human origin strain stands for the A/Cambodia/408008/2005 strain.*last day of the corresponding experiment at which samples could be collected and tested.</p

Viral matrix (M) gene expression copy number normalized to β-actin gene expression (10⁵ copies) by quantitative RT-PCR in influenza virus infected mouse bone marrow derived macrophages.

<p>Matrix gene mRNA copy number was assayed 3 h, 6 h and 24 h post-infection and normalized to those of β-actin mRNA in the corresponding sample. Means of triplicate assays are shown with standard error. Asterisk indicates statistical difference (<i>p</i><0.05).</p

Cytokine and chemokine secretion from mouse bone marrow derived macrophages after influenza A virus infection.

<p>(A) TNF-α (B) IP-10 protein secreted by the mouse bone marrow derived macrophages after influenza A viruses infection (as denoted in legend). Mean and standard error of duplicate assays are shown. All influenza A virus infected mouse macrophages secrete significantly higher concentration of TNF-α than mock infected cells (<i>p</i><0.05).</p

Virus titer detected in the supernatant of influenza A virus infected mouse bone marrow derived macrophages.

<p>Virus titer of various (A) influenza H1N1, and (B) H5N1 influenza viruses was determined at 3, 24, 48 and 72 h post-influenza virus infection of mouse bone marrow derived macrophages. Means and standard error of triplicate assays were shown. Dotted line represents the lowest detection limit of the TCID₅₀ assay. The thermal inactivation (serial dilution of influenza virus was incubated in the cell-free culture medium alone at the corresponding time points) curves (dotted line) of influenza H1N1 and H5N1 viruses at 37°C were determined from culture wells without macrophages.</p

Cell characterization and lectin profile of the mouse bone marrow derived macrophages.

<p>Histogram showing the percentage of positive stained mouse bone marrow derived macrophages by flow cytometry (open peak-blue line). Isotype control (open peak-black line) and non-stained cells as negative control (shaded peak) of bone marrow derived macrophages stained with (A) CD14 and (B) F4/80. Lectin immune-staining assay to determine the sialic acid (SA) distribution on mouse bone marrow derived macrophages. (C) <i>Maackia amurensis</i> lectin (MAA) conjugated with FITC (the lectin that binds SA-α2,3Gal linked sialic acid) and (D) with <i>Sambucus nigra</i> lectin (SNA) conjugated with FITC (the lectin that binds SA-α2,6GalNAc).</p

Lectin binding: MAL-I binds to Sia-α2-3Galβ1-4GlcNAc (Panel A, D, G and J), MAL-II binds to Sia-α2-3Galβ1-3GalNAc (Panel B, E, H and K) and SNA binds to Sia-α2-6-linkage (Panel C, F, I and L) to determine the Sias distribution on the ud- and wd-NHBE cells.

<p>MAL-I, MAL-II and SNA bindings presented on the (A-C) <i>en face</i> staining of ud-NHBE, (D-F) cross-section staining of ud-NHBE, (G-I) cross-section staining of wd-NHBE cells <i>in vitro</i> cultures and (J-L) the human bronchial biopsy in reddish brown.</p