Expression of chMDA5(1-483) induces type I IFN.

<p>The effect of chMDA5(1-483) expression on the activation of the chIFN-β promoter (a) and on type I IFN secretion (b) was analyzed in the DF-1 fibroblast cell line (a) and in the HD-11 macrophage-like cell line (b), respectively. DF-1 cells were transfected with the reporter plasmids for measuring chIFN-β promoter activity and with the indicated expression constructs (a). The firefly and <i>Renilla</i> luciferase activities were determined 24 h later. The IFN-β promoter-induced firefly luciferase activity was normalized with the corresponding <i>Renilla</i> luciferase activity and expressed as fold induction compared to cells transfected with the empty expression plasmid (Vector). The bars represent the mean values of five independent transfections with the error bars showing the standard deviations. The graph is representative of two independent experiments. For type I IFN induction, HD-11 cells were transfected with the indicated plasmids (b). Eighteen hours later, the cell supernatants were assayed for type I IFN using the bioassay as described. The bars represent the mean values of six independent transfections with the error bars showing the standard deviations.</p

Quantification of viral RNA in cloacal swabs from animals vaccinated with 25 µg of DNA.

<p>Abbreviations: dpc, days post-challenge; -, PCR negative; RNA copies per 100 µl swab, (+) <10³,+<10⁴,++<10⁵,+++<10⁶,++++<10⁷,+++++<10⁸;</p>†<p>animal dead;</p>††<p>animal euthanized.</p

Effect of chMDA5(1-483) co-expression on HA-specific antibody responses elicited by HA DNA vaccination.

<p>Groups of 6 chickens were immunized with a plasmid DNA mixture containing equal amounts of the plasmids for HA expression and for chMDA5(1-483) expression. Doses of 25 µg or 2.5 µg of each plasmid DNA per animal were applied to two groups of 6 animals/dose, as indicated. The same two doses of plasmid DNA for HA expression alone mixed with an equal amount of empty expression plasmid were applied to two other groups of 6 chickens. Two control groups of 3 chickens each received 25 µg or 2.5 µg of empty expression plasmid each (Vector). Two immunizations were applied at an interval of 23 days. 20 days after the second vaccination (<i>i.e.</i> at 43 dpi), the chicken sera were tested for the presence of HA-specific antibodies. H5-specific antibodies were detected using a commercial competition ELISA. The reactivity of HA antibodies was calculated according the formula 100-[OD(Probe)/OD(Negative)x100] (a). Alternatively, the anti-HA IgY titers were determined in an indirect ELISA using immobilized recombinant HA protein and represented as reciprocal of the highest dilution that yielded an OD greater than 2.1 x of parallel preimmune serum samples (b). The HI titer was determined as the highest serum dilution resulting in complete inhibition of aggregation of chicken red blood cells caused by 8 HAU of Vac-1/04 (H5N1) virus (c). The neutralizing antibody titers in serum samples were determined with Vac-1/04 virus and plotted as reciprocal of the highest dilution resulting in 50% virus neutralization (d). The HA-specific IgA antibody content in the serum was measured by ELISA. Results are shown as absolute OD values from which the unspecific background values were subtracted (e). Each symbol represents an individual animal. The * indicates statistical significant differences calculated with the students t-test (p<0.05).</p

Schematic overview of the plasmid constructs used for DNA vaccination.

<p>The pcDNA6-V5-His B plasmid backbone (Vector) carrying a cytomegalovirus immediate early promoter (P_CMV), a multiple cloning site (MCS) and the transcription termination and polyadenylation sequence (pA) was used for all protein expression constructs. The monocistronic expression plasmids pcDNA6-HA and pcDNA6-chMDA5(1-483) carry the HA gene of the H5N1 AIV Yamaguchi-7/04 and a cassette encoding the N-terminal 483 amino acids of the chicken MDA5, respectively. In the bicistronic expression plasmids pcDNA6-HA-IRES-eGFP and pcDNA6-HA-chMDA5(1-483), the IRES of the encephalomyocarditis virus was used to separate the HA gene from the eGFP and the chMDA5(1-483) gene, respectively. The plasmid pcDNA6-HA-IRES carries an empty MCS downstream of the IRES.</p

Quantification of viral RNA in tracheal swabs from animals vaccinated with 25 µg of DNA.

<p>Abbreviations: dpc, days post-challenge; -, PCR negative; RNA copies per 100 µl swab, (+) <10³,+<10⁴,++<10⁵,+++<10⁶,++++<10⁷,+++++<10⁸;</p>†<p>animal dead;</p>††<p>animal euthanized.</p

Effect of chMDA5(1-483) co-expressed with HA on clinical symptoms and survival after challenge with HPAIV H5N1.

<p>The chickens received two intramuscular immunizations with 25 µg (a and b) or 2.5 µg (c and d) of the indicated plasmid DNA at 23 days interval. After another 23 days (or at 46 dpi), the animals were challenged by intratracheal administration of the HPAIV Yamaguchi-7/04. The chickens were observed daily for clinical signs for a period of 9 days after the challenge infection. The daily clinical index was monitored as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049952#pone.0049952-Veits1" target="_blank">[58]</a> with minor modifications: healthy (0), reduced activity (0.25), slightly ill (0.5), ill (1), severely ill (2), severely ill and euthanized (2.5) or dead (3). The daily clinical index is represented as the mean value of all chickens per group (a and c). The number of surviving animals is plotted against the time (in days) after the challenge (b and d). Note that the two groups (25 µg and 2.5 µg dose) vaccinated with control vector plasmid (Vector) consisted of only three animals each.</p