Article Figures & Data
Figures
- FIG. 1.
Functional analysis of the influenza virus reporter amplicon with a canine polymerase I promoter in MDCK cells. (A) The negative-sense Renilla luciferase [R-LUC(−)] coding region is flanked by modified noncoding regions (NCR; hatched boxes) from the nucleoprotein gene of A/WSN/33 virus (24, 30). The sequences of the canine POL-I promoter and the canine Pol-I terminator (k9POL-I and k9TI, respectively; gray box) were fused upstream to the 5′ NCR or downstream to the 3′ NCR, respectively. (B) MDCK cells were cotransfected with reporter plasmid pk9POLI-RLuc and influenza virus RNP-expressing plasmids or control plasmid. RLU, relative light units. (C) MDCK cells transfected with pk9POLI-RLuc plasmid for 24 h were infected with influenza viruses at an MOI of 0.001. Luciferase activities in whole-cell lysates collected at 24 h postinfection are shown as the average of luciferase activity of cells from three independent wells. The values shown are the Renilla firefly activities from 104 cells after normalization using firefly luciferase expression from a cotransfected plasmid to account for variation in transfection efficiency. Error bars depict standard errors, and brackets denote P values from Student's t test: *, P > 0.9; **, P < 0.05; and ***, P < 0.005.
- FIG. 2.
Induction of luciferase activity in the reporter Luc9.1 cells upon influenza virus infection. (A) Specificity of luciferase activation in Luc9.1 reporter cells. Luc9.1 cells were infected with A/Ohio/83 (H1N1), A/Wisconsin/67/05 (H3N2), or B/Jiangsu/10/03 virus at an MOI of 0.1. (B) Detection limits of Luc9.1 cells upon influenza virus infection. Luc9.1 cells were inoculated with the indicated amounts of infectious A/Ohio/83 virus. Luciferase activity in each culture was measured in lysates harvested at 24 h postinfection. Data represent average luciferase activity of 104 cells from three independent wells. Error bars depict standard error, and brackets denote P values from Student's t test. *, P < 0.005.
- FIG. 3.
Time course of luciferase activity induction in the reporter Luc9.1 cells upon influenza virus infection. Luc9.1 cells were inoculated with A/Ohio/83(H1N1), A/Wisconsin/67/05 (H3N2), or B/Jiangsu/10/03 at an MOI of 0.001. Renilla luciferase activity was measured at different times after infection. Data represent normalized luciferase activity of 104 cells from three independent wells.
- FIG. 4.
Luciferase activity in Luc9.1 cells upon infection with seasonal, avian and 2009 pandemic H1N1 influenza A viruses. (A) Luc9.1 cells were infected with the seasonal H1N1, H3N2, pandemic H1N1, or virus isolated from animal species at an MOI of 0.01. Renilla luciferase activity was measured at 24 h postinfection. Error bars depict standard error, and brackets denote P values from Student's t test: *, P < 0.001. (B) Luc9.1 cells were inoculated with highly pathogenic H5N1 (A/Vietnam/1203/04, A/Vietnam/JP 12-2/05, and A/Hong Kong/213/03) at an MOI of 0.01. Renilla luciferase activity was measured at 24 h postinfection. Error bars depict standard error, and brackets denote significance per Student's t test. *, P < 0.05.
- FIG. 5.
Performance characteristics of the Luc9.1 cells in a virus neutralization assay. Two-fold dilutions of convalescent A/Sydney/5/97 ferret antiserum were incubated with 1,000 PFU of H1N1 (A/Ohio/83) or H3N2 (A/Sydney/5/97) virus. Intracellular Renilla luciferase was measured 24 h after infection. Data represent normalized luciferase activity of 104cells from three independent wells. Error bars depict standard error, and brackets denote P values from Student's t test. *, P < 0.05.
- FIG. 6.
Performance characteristics of the Luc9.1 cells in antiviral drug assays. (A) Amantadine-resistant virus (A/Wisconsin/67/05) and amantadine-sensitive viruses (A/Sydney/5/97 and A/Ohio/83) were incubated with 5 μM amantadine and added to Luc9.1 cells. Luciferase activity was measured at 24 h postinfection. (B and C) A/Ohio/83 virus was incubated with different concentrations of amantadine and added to Luc9.1 cells (B) and parental MDCK cells (C). Luciferase activity was measured 24 h postinfection from Luc9.1 cells. MDCK cell supernatants were collected 48 h later for HA titration. Data represent normalized luciferase activity of 104 cells from three independent wells. Error bars depict standard error, and brackets denote P values from Student's t test. *, P < 0.001.
