An adenovirus vector-mediated reverse genetics system for influenza A virus generation

Abstract

Plasmid-based reverse genetics systems allow the generation of influenza A virus entirely from cloned cDNA. However, since the efficiency of virus generation is dependent on the plasmid transfection efficiency of cells, virus generation is difficult in cells approved for vaccine production that have low transfection efficiencies (e.g., Vero cells). Here we established an alternative reverse genetics system for influenza virus generation by using an adenovirus vector (AdV) which achieves highly efficient gene transfer independent of cell transfection efficiency. This AdV-mediated reverse genetics system will be useful for generating vaccine seed strains and for basic influenza virus studies.

FIG. 1.

Schematic diagrams of the transcription cassettes of pPolI and AdVs for reporter vRNA synthesis. In pPolI-GFP (14), the 3′ noncoding region of NP vRNA (3′ NCR), the GFP open reading frame in the negative sense, and the 5′ noncoding region of NP vRNA (5′ NCR) were inserted between the PolI promoter (P_PolI) and the PolI terminator (T_PolI). In cells transfected with pPolI-GFP, the reporter vRNA containing the GFP gene is synthesized by cellular PolI (A). AdV/PolI-GFP possessed the same transcription cassette of pPolI-GFP for reporter vRNA synthesis (B). The vRNA transcriptional region in AdV/CMV-PolI-GFP was flanked by the human cytomegalovirus immediate-early promoter (P_CMV) and the herpes simplex virus thymidine kinase polyadenylation signal (TK pA). In cells transduced with AdV/CMV-PolI-GFP, the reporter vRNA and mRNA containing the GFP genes are synthesized by cellular PolI and PolII, respectively. The backbone of the adenovirus clones (Ad) was the genome of adenovirus type 5 with E1 and E3 deleted. The transcriptional initiation site and orientation of the GFP gene are indicated by the white arrow. All of the recombinant replication-incompetent AdVs used in this study were produced by the ViraPower Adenoviral Expression System (Invitrogen) according to the manufacturer's instructions.

FIG. 2.

GFP expression in Vero cells transduced with AdVs for reporter vRNA synthesis. Vero cells were transduced with AdV/PolI-GFP (A to C) and AdV/CMV-PolI-GFP (D and E). Simultaneously, the cells were transfected with plasmids (B) or transduced with AdVs (C and E) for the expression of the polymerase subunits (PB2, PB1, and PA) and NP. Forty-eight hours later, GFP expression was examined by fluorescence microscopy. In each experiment, each AdV was transduced at an MOI of 50. The image in panel D was taken with a 10-fold longer exposure time than those in the other panels. Scale bars, 200 μm.

FIG. 3.

Comparison of the virus generation efficiency of plasmid transfection systems and AdV transduction systems. Vero cells were transfected with 12 plasmids (11) or 3 plasmids (pTM-PolI-WSN-All, pC-PolII-WSN-PB2-PB1-PA, and pCAWS-NP) (10) or transduced with 12 AdVs (AdV/PolI-PB2, -PB1, -PA, -HA, -NP, -NA, -M, and -NS and AdV/CMV-PB2, -PB1, -PA, and -NP) or 8 AdVs (AdV/CMV-PolI-PB2, -PB1, -PA, -HA, -NP, -NA, -M, and -NS). Seventy-two hours later, virus titers in culture supernatant were determined by plaque assay on MDCK cells. The virus titer detection limit of our system was 5 PFU/ml. The results of three independent experiments (Exp.) are shown.