Analysis of host gene involvement in major cellular pathways.

<p>A: A549 cells were reverse cotransfected with CRE/CREB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33) B: A549 cells were reversed cotransfected with NF-κB reporter plasmid or the appropriate control plasmid and 100 nM of the novel siRNA. After 24 h incubation, the transfection media was replaced with culture media. Cells were mock infected or infected with A/WSN/33 at an MOI of 0.001 the following day. After 24 h, culture supernatant was analyzed for luciferase expression. Luciferase units were normalized to Renilla expression. * p<0.05, ** p<0.01 compared to siNEG control (mock), # p<0.05, ## p<0.01 compared to siNEG control (A/WSN/33). Data is representative of three independent experiments.</p

RNAi of individual host protease genes down-regulates replication of a clinical influenza isolate.

<p>A549 cells were reverse transfected with 100 nM of the novel siRNA targeting siADAMTS7, siCPE, siDPP3, siMST1, and siPRSS12. After 48 hours, cells were infected with A/New Caledonia/20/99 at an MOI of 0.1 in the presence of 1 ug/ml TPCK-trypsin. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID₅₀. Data is expressed as TCID₅₀/ml. Data is representative of two independent experiments. (*p<0.05 vs. siNEG).</p

RNAi of DPP3 (siDPP3) inhibits influenza replication by modulation of apoptotic genes.

<p>A549 cells were reverse transfected with 50 nM of siDPP3 or siNEG. After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. After 18 hours of infection, cellular RNA was isolated and apoptosis gene expression profiles were determined by array. Gene expression was normalized to GAPDH levels. Silencing DPP3 resulted in upregulated levels of the pro-apoptotic genes BCL2L10, TNFSF10, TNFSF25 and TNFSF8. Data is representative of three independent experiments. * p<0.05.</p

Human protease gene hits.

*<p>: Refers to the pool of four siRNAs used in the primary screen.</p>#<p>: Refers to the individual siRNA used for the validation step.</p

RNAi of 5 host protease genes down-regulated influenza virus replication.

<p>A: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cytotoxicity was determined by adenylate kinase (AK) release. Cells treated with the siTOX control were considered 100% cytotoxic and all values were normalized to siTOX. RLU = relative luciferase units. * p<0.05 vs siNEG, ** p<0.01 vs siNEG, ***p<0.005 vs siNEG; siTOX vs all samples: p<0.001 (not shown). Line shows 20% of siTOX control. B: A549 cells were reverse transfected with 50 nM of siRNA (SMARTpool) specific for the indicated genes (ADAMTS7, CPE, DPP3, MST1, PRSS12). After 48 hours, cells were infected with A/WSN/33 at an MOI of 0.001. 48 hours post-infection, cells were fixed in 4% formaldehyde and stained with an anti-NP (green) monoclonal antibody followed by counterstain with DAPI (blue.) Positive (+) control: siMEK, negative (−) control: siNEG. Magnification is 20× (bar is 100 microns). C: Cells were transfected with 100 nM of a novel siRNA targeting a different seed site from the SMARTpool used in the primary screen and infected as in B. After 48 hours of infection, cellular supernatant was tested for infectious virus production by a modified TCID₅₀. Data is expressed as TCID₅₀/ml. Data is representative of two independent experiments. (*p<0.05 vs siNEG).</p

Development of a competitive astrovirus ELISA.

<p>A) Rabbit anti-HAstV-1 or TAstV-2 polyclonal antibodies were pre-incubated with PBS (no treatment) or purified recombinant HAstV-1 or TAstV-2 capsid proteins then tested for binding to bound HAstV-1 or TAstV-2 capsid protein by ELISA. *p<0.05. B) Specificity of the rabbit polyclonal antibodies. Samples were run in at least duplicate and data are shown as mean values. Error bars indicate SD.</p

Specificity of the human sera to HAstV-1 and TAstV-2 capsid proteins.

<p>A) HAstV-1 positive sera were pre-incubated with PBS (no treatment) or HAstV-1 or TAstV-2 capsid proteins and binding to HAstV-1 capsid protein assessed by ELISA. B) TAstV-2 positive sera were pre-incubated with PBS (no treatment) or HAstV-1 or TAstV-2 capsid proteins and binding to TAstV-2 capsid protein assessed by ELISA. C) TAstV-2 positive and negative sera were pre-incubated with PBS (no treatment) or chicken astrovirus and binding to TAstV-2 capsid protein assessed by ELISA. A plus sign denotes serum sample was positive for indicated virus, and a minus sign denotes serum sample was negative for the indicated virus. Samples were tested in duplicate and data shown are mean values; error bars indicate SD. *p<0.05.</p

Serologic results of TAstV-2 and HAstV-1 antibody testing.

1<p>4/4 samples positive to 1∶100 dilution.</p>2<p>Of 12 TAstV-2 positive samples tested, 8.3% positive to 1∶1000, 75% positive to 1∶10000, and 16.7% positive to 1∶100000.</p

Serologic and demographic results of Cohort C.

1<p>p-value associated with pearson Chi-square test.</p

Effect of miRNA inhibition on influenza replication.

<p>A549 cells were treated with the appropriate miRNA inhibitor (25 nM) for 48 hours, followed by infection with A/WSN/33 (MOI = 0.001). Cellular supernatant was tested for infectious virus production by a modified TCID₅₀ 48 hpi. Data is expressed as TCID₅₀/ml and is representative of two independent experiments.</p