293 T and Vero cells were maintained in Dulbecco’s modified Eagle minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). CEFs were prepared from 11-day-old embryonated chicken eggs by trypsin digestion and stored in liquid nitrogen. CEF and MDCK cells were maintained in DMEM containing 10% or 5% FBS, respectively.
A standard replicon assay was performed as previously described [41, 42, 77]. Luciferase activity was measured using the Dual-Glo luciferase assay kit and a GloMax 96 Microplate Luminometer (Promega, Fitchburg, USA) after transfection of 0.1 μg each of pCPB2, pCPB1, pCPA, pCNP, and ppolINSluc and 0.01 μg of pRLSV40 (Promega) using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). The PB2, PB1, PA, and NP genes of influenza A/Cambodia/P096/2005 (H5N1)  and A/WSN/33 (H1N1)  were PCR-amplified using specific oligonucleotides and cloned into pcDNA3.1(+) (Invitrogen), resulting in pcCamPB2wt, pcCamPB1, pcCamPA, pcCamNP, pcWSNPB2, pcWSNPB1, pcWSNPA, and pcWSNNP, respectively. Only data in which the variation of the Renilla luciferase activity was less than 3-fold variations were used. The oligonucleotide sequence information will be made available upon request.
Expression, purification, and in vitro transcription of the influenza virus RdRp
Expression, purification, and in vitro transcription of the influenza virus RdRp were performed as previously described [42, 77, 78]. Briefly, 100 nM influenza RdRp was incubated in 50 mM Tris–HCl (pH 8.0), 8 mM MgCl2, 150 mM NaCl, 2 mM DTT, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.05 mM [α-32P] UTP, 0.1 mM ApG or 0.01 mg/mL globin mRNA, 2000 U/mL RNase inhibitor, and 200 nM v84 and c84 model template RNA at 25°C for 90 min. The product was analyzed by PAGE on 6% gels containing 8 M urea and the images analyzed with a Typhoon Trio plus (GE Healthcare, Bucks, UK).
Model RNA templates
v84 and c84 model RNA templates were prepared as previously reported .
Proteins were blotted onto nitrocellulose membranes (Millipore, Billerica, USA) by semi-dry electroblotting (Bio-Rad, Hercules, USA) after SDS-PAGE on 10% gels. The blotted membranes were blocked with 10% skim milk in 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.02% Tween 20 (TBST), and western blotting was performed using rabbit anti-PB1, -PB2, -PA , and -PR8 (1:1,000 each) and anti-actin (1:100) antibodies as the primary antibodies. The membranes were then incubated with alkaline phosphatase-conjugated anti-rabbit IgG (1:7,500) or anti-mouse IgG (1:7,500), and the positions of the bound antibodies were visualized with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Reconstitution of influenza virus and plaque-formation assay
pHH21 and the influenza virus 12-plasmid reconstitution system of WSN were kindly provided by Dr. Hobom and Dr. Kawaoka . The H5N1 A/Cambodia/P0322095/2005  PA sequence was inserted into ppolI-WSN-PA, resulting in ppolI-Cam-PA. The influenza virus WSN strain (WSN) and WSN carrying H5N1 Cambodia PA (C-PA) were reconstituted by co-transfection of 293 T cells with ppolI-WSN-PB2, ppolI-WSN-PB1, ppolI-WSN-PA or ppolI-Cam-PA, ppolI-WSN-HA, ppolI-WSN-NP, ppolI-WSN-NA, ppolI-WSN-M, and ppolI-WSN-NS with pCPB2, pCPB1, pCPA, and pCNP [10, 56] using Lipofectamine 2000. The reconstituted viruses were recovered from the culture media 72 hr post-transfection. The viruses were plaque-purified, amplified, and titered on MDCK cells by a plaque-formation assay and stored at −80°C. The genome sequences of all viruses were determined by RT-PCR. The primer sequences used will be made available upon request.
Virus growth assay
MDCK, CEF, and Vero cells in 3.5-cm dishes (21 dishes of MDCK cells and CEFs, 27 dishes of Vero cells per a virus) were infected with WSN and C-PA at an MOI of 0.01 at 37°C for 1 hr (multi-step growth assay). The cells were washed 3× with phosphate-buffered saline (PBS) and incubated with 2 mL of DMEM containing 2% FBS (without trypsin) at 37°C, as WSN replicates without trypsin . All of the supernatants of 3 dishes of each cell were taken 6, 12, 24, 36, 48, and 60 hr (and also at 72 and 90 hr for Vero cells) after infection and stored at −80°C. Virus growth was also tested in MDCK and Vero cells after infection at an MOI of 5 (single-step growth assay). Supernatants were harvested 2, 4, 6, 8, 10, and 12 hr after infection. The viruses in the supernatants were titered on MDCK cells by a plaque-formation assay.
Groups of 4 6-week-old female BALB/c mice (Sino-British Laboratory Animal, Shanghai, China) were anesthetized with ether and inoculated with 105, 104, or 103 PFU of WSN or 106, 105, 104, or 103 PFU of C-PA in a volume of 50 μL by nasal dropping. Four mice were inoculated with 50 μL of PBS as a mock-infection control. The survival rates were monitored daily and the 50% lethal doses (LD50s) calculated .
Six-week-old female BALB/c mice were anesthetized with ether and inoculated with 105 PFU of WSN or C-PA in 50 μL by nasal dropping. Mice were inoculated with 50 μL of PBS as a mock-infection control. A non-treated mouse was used as a non-treated control. The mock-infected and infected mice were sacrificed by cervical dislocation under anesthesia on days 1, 2, 3, and 4 after infection, and their lungs were removed and fixed with 3.5% formalin/PBS at 25°C for 2 days. The lungs were embedded in paraffin blocks, sectioned at 4-μm thickness, and stained with hematoxylin and eosin (HE).
Cells were placed on cover slips in 24-well plates and infected with WSN or C-PA at an MOI of 0.01. Eight, 9, 10, 11, 12, 13, 14, 15, 16, and 24 hr after infection, cells were fixed with 1% formalin/PBS. The TUNEL assay was performed using the DeadEndTM Fluorometric TUNEL system according to the company’s instructions. The samples were observed using a fluorescence microscope (Leica DM IRB, Leica, Wetzlar, Germany), and the numbers of TUNEL positive cells in 3 random fields were counted.
MDCK cells were plated in 10-cm-diameter plates and infected with WSN or C-PA at an MOI of 0.01. 10 hr after infection, the cells were harvested and the activities of caspases 3, 8, and 9 measured using the Caspase-3/CPP32 Fluorometric Assay kit, the Caspase 8/FLICE Fluorometric Assay kit, and the Caspase 9 Fluorometric Assay kit (Biovision, Inc., Milpitas, USA) according to the manufacturer’s instructions.
Interferon induction was analyzed by measuring the luciferase activity of cells transfected with p-125Luc, which was kindly provided by Dr. Fujita . 293 T cells were transfected with p-125Luc (1 μg) and pRLSV40 (100 ng). Four hours post-transfection, the cells were infected with WSN or C-PA at an MOI of 0.01. The interferon promoter activity was measured as the luciferase activity using the Dual-Glo luciferase assay kit and a GloMax 96 Microplate Luminometer (Promega) and normalized as the firefly luciferase/Renilla luciferase activity ratio.
Chemicals and radioisotopes
Non-radiolabeled nucleotides were purchased from GE Healthcare, [α-32P]UTP from New England Nuclear (PerkinElmer Life Sciences, Waltham, USA), and T7 RNA polymerase, T4 nucleotide kinase, oligonucleotides, human placental RNase inhibitor, and restriction enzymes from Takara (Dalian, China). The RPAIII Ribonuclease Protection Assay Kit was purchased from Ambion (Austin, USA). Anti-actin antibodies were purchased from Sigma Aldrich (St. Louis, USA). The Dual-Glo luciferase assay kit, DeadEndTM Fluorometric TUNEL system, alkaline phosphatase-conjugated anti-rabbit and anti-mouse IgG’s, NBT, and BCIP were purchased from Promega. DMEM, FBS, Lipofectamine 2000, and Trizol reagent were purchased from Invitrogen.
The statistical significance levels of the data were evaluated by Student’s t-test, with p < 0.05 indicating statistical significance.