Figure 1.
Replication and IFN-β induction by human and avian H5N1 strains in human cells.
(A, B) A549 cells were infected at a multiplicity of 0.01 (A) or 1 (B) with the highly-pathogenic human H5N1 IAV isolates A/Hong Kong/156/1997 (HK/97) and A/Thailand/1 (KAN-1)/2004 (Thai/04), the avian H5N1 strains A/chicken/Indonesia/R132/2004 (Ch/Ind), A/duck/Vietnam/TG24-O1/2005 (Duck/VN) and A/common buzzard/Berlin/1/2006 (Buzz/Bln) as well as with the prototypic seasonal A/Panama/2007/99 (Pan/99) H3N2 virus and its mutant variant with a deleted NS1 gene (Pan-delNS1; shown only in panel A). Supernatant of infected cell cultures was sampled at the indicated time points and viral titers were determined by plaque assay. Mean data+SEM of at least three independent experiments is shown. (C) Immunoblot analysis of lysates from A549 cells infected at MOI = 1 using a viral NP-specific antibody at the indicated time points post infection (p.i.). Relative intensities of NP bands normalized to actin controls are depicted in comparison to Thai/04, which was arbitrarily set as 100%. (D, E) Concentrations of IFN-β in cell culture supernatants of A549 cells infected with the indicated viruses at low (MOI = 0,01, panel D) or high multiplicity (MOI = 1, panel E) determined via ELISA at 24 and 48 hpi (D) or at 16 hpi (E). Mean data of at least three independent experiments +/– SEM is shown. Symbols indicate significant differences between given results with p<0.05 (*), p<0.01 (**), or p<0.001(***), respectively, as determined by unpaired two-tailed t-tests.
Figure 2.
H5N1 virus growth is sensitive to IFN-α/β.
(A) A549 lung epithelial cells were treated with 500 U/ml recombinant IFN-α before and during infection with the indicated virus strains (MOI = 0.01). Viral titers were determined by plaque assay. Mean titers at 24 and 48 hours post infection of at least 3 independent experiments+SEM are presented. For comparison, the graph also depicts virus titers determined in untreated A549 cells at 24 and 48 hpi. shown in Figure 1A. Symbols indicate significant differences between given individual results with p<0.05 (*) or p<0.01 (**), respectively. (B) Virus growth of human and avian H5N1 isolates, Pan/99 (H3N2) and its deltaNS1 variant on IFN-α/β-deficient Vero cells infected at a MOI of 0.01. Samples of the infected cell cultures supernatants were taken at the indicated time-points and viral titers were determined by plaque assay (mean data+SEM; N ≥3).
Figure 3.
IFN-α/β secretion in response to H5N1 infection depends on RIG-I expression.
A549 cells, transfected with either RIG-I-specific (+, open columns) or control (–, solid columns) siRNA were infected with the indicated H5N1 and the Pan/99 (H3N2) derived viruses (MOI = 1). IFN-β concentrations were determined via ELISA (Fujirebio, Japan) in cell culture supernatants taken 16 h post infection (upper panel) and RIG-I expression was determined by immunoblotting (lower panel). Mean data +/– SEM of four independent experiments and a representative immunoblot is shown.
Figure 4.
Replication of reassortant Pan/99 (H3N2)×H5N1-NS viruses in lung epithelial cells.
(A) A549 cells were infected with the indicated Pan/99 (H3N2) wild-type and reassortant viruses carrying a H5N1-NS segment (MOI = 0.01). Aliquots of the supernatants were taken at the indicated time points and viral titers were determined by plaque titration. Mean data of at least two independent experiments+SEM are shown. (B) Virus growth on IFN-α treated A549 cells (500 IU/ml, 6 h before infection (MOI = 0.01) and during virus growth) was determined as described in panel A. Shown are the average results of two independent experiments+SEM.
Figure 5.
H5N1 NS1 proteins inhibit the induction of type I IFN in human cells.
(A) IFN-β concentrations of supernatants sampled from A549 cell cultures infected with the Pan99 WT and different NS reassortant viruses (H3N2) (MOI = 0.01) at 24 and 48 hrs p.i., (N≥2+/–SEM). (B) Human 293T cells were co-transfected with plasmids expressing the H5N1- or Pan/99 (H3N2)-derived NS segments or empty vector, the human IFN-β promoter reporter plasmid p125-Luc and pRL-TK-Luc to control for transfection efficiency. Subsequently, cells were infected for 16 h with Pan-delNS1 to stimulate the IFN-β promoter before luciferase reporter activity was determined in cell lysates. IFN-β promoter activation in infected cells is shown as x-fold stimulation of firefly luciferase activity compared to transfected and mock infected cells. Mean data of three independent experiments is shown +/– SD. (C) Immunblot detection of viral NP after Pan-delNS1 (H3N2) infection confirmed equal stimulation of the transfected cells analyzed in panel B.
Table 1.
Differences in H5N1 influenza virus proteins*.
Table 2.
Common differences between examined human and avian H5N1 strains*.