Schematic representation of the plasmid-based rescue system for influenza A virus. The pGT-h set of protein expression plasmids was constructed by inserting the open reading frames of PB1, PB2, PA, and NP proteins into the BclI cloning site of the pGT-h plasmid (2). The PB1 and PA genes were derived from influenza A/WSN/33 virus. The PB2 and NP genes were derived from influenza A/PR/8/34 virus. The viral genomic sequences of influenza A/WSN/33 virus were cloned into pUC18- or pUC19-based plasmids between a truncated human RNA Pol I promoter (nt −250 to −1) (19) and sequences of the hepatitis delta virus ribozyme in an analogous way as described for pPOLI-CAT-RT (28). Genetic tags were inserted into the HA- and NA-encoding plasmids by using conventional mutagenic techniques. For viral rescue, 5 μg of each of the polymerase protein expression plasmids (pGT-h-PB1, pGT-h-PB2, and pGT-h-PA), 10 μg of the NP-expressing pGT-h-NP, and 3 μg of each of the eight vRNA-coding transcription plasmids (pPOLI-PB2-RT, pPOLI-PB1-RT, pPOLI-PA-RT, pPOLI-HA-RT, pPOLI-NP-RT, pPOLI-NA-RT, pPOLI-M-RT, and pPOLI-NS-RT) were diluted to a concentration of 0.1 μg/μl in 20 mM HEPES buffer (pH 7.5). The DNA solution was added to diluted DOTAP liposomal transfection reagent (Boehringer) containing 240 μl of DOTAP and 720 μl of 20 mM HEPES buffer (pH 7.5). The transfection mixture was incubated at room temperature for 15 min, mixed with 6.5 ml of MEM containing 0.5% fetal calf serum, 0.3% bovine serum albumin, penicillin, and streptomycin and added to near-confluent Vero cells washed with phosphate-buffered saline in 8.5-cm dishes (about 10⁷ cells). At 24 h after transfection, the transfection mixture was removed and the cells were incubated with 8 ml of fresh medium, which was replaced daily for 4 days. The harvested medium from transfected dishes was screened for rescued influenza virus by plaquing and amplification on MDBK cells. POL I, truncated human RNA polymerase I promoter; R, genomic hepatitis virus ribozyme; MLP, adenovirus type 2 major late promoter; pA, polyadenylation sequence from SV40.

Demonstration of the presence of genetic tags in the HA and NA vRNA segments of the rescued virus by RT-PCR and restriction enzyme analysis. vRNA of the rescued virus was isolated from medium of infected MDBK cells. One hundred microliters of the medium was treated with 5 U of RNase-free DNase to remove any residual plasmid DNA carried over. After 15 min at 37°C, vRNA was isolated by using the RNeasy Mini Kit (Qiagen), following the manufacturer’s instructions. vRNA from authentic wild-type A/WSN/33 virus was isolated from purified virus as described previously (10). The first 149 nt at the 3′ end of the HA vRNA were amplified by RT-PCR using oligonucleotide primers 5′-GCGCTCTAGAGCAAAAGCAGGGGAAAATAA-3′ (corresponding to nt 1 to 21) and 5′-CGCGAAGCTTCTCGAATATTGTGTCAAC-3′ (corresponding to nt 129 to 149), resulting in a 165-nt-long PCR product. To amplify the sequence containing the genetic tag from the NA segment, primers 5′-TGGACTAGTGGGAGCATCAT-3′ (corresponding to nt 1280 to 1309) and 5′-GAACAAACTACTTGTCAATGGT-3′ (corresponding to nt 1367 to 1388) were used in RT-PCR to produce a 108-nt PCR product. The HA- and NA-specific PCR products were incubated for 2 h at 37°C in the presence (+) or absence (−) of 10 U of SpeI and SacI restriction enzymes, respectively. Samples were analyzed on 16% polyacrylamide gels and stained with ethidium bromide. Lanes: 1, DNA size markers (sizes in nucleotides are indicated); 2, 3, 7, and 8, PCR products from the rescued virus; 4 and 9, control reactions omitting reverse transcriptase; 5, 6, 10, and 11, PCR products from the authentic wild-type A/WSN virus.