Influenza A/California/12/2009(H1N1) was kindly provided by the Laboratory of Virology, Chungbuk National University and wild-type novel influenza A/H1N1 was obtained from the Department of Laboratory Medicine, National Health Insurance Corporation Ilsan Hospital.
A host cell and chicken embryos
A host cell, MDCK, for the culture of the viruses from nasal swabs and aspirates was kindly provided by the laboratory of Professor Chan-Hee Lee. Chicken embryos were purchased from an egg store in Korea (Ochang Mart, Ochang, Republic of Korea).
For the culture of Influenza from the aspirates of nasal swabs, MDCK cells were cultured in DMEM (Invitrogen Corporation, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS, Invitrogen Corporation) at 37°C and 5% CO2. Before infection, MDCK cells were trypsinized in 1 × 107 cells per T75 flasks (Nalge Nunc International, Rochester, NY). After seeding in 10 mL DMEM plus 5% FBS at 80-90% confluency, plated cells were rinsed with phosphate-buffered saline (PBS, Invitrogen Corporation). Suspended specimens in the flasks were gently shaken for two hours for effective mixing. Cell infections were observed until 80-90% of the cells were floating or lightly attached to the T75 flask (typically 4-5 days post-infection). At that time, harvesting and storage of viral supernatant was performed.
The titer of the influenza A/H1N1 virus was determined by HA assay. First, U-bottom 96 well plates (Nalge Nunc International) were prepared and 50 μL of PBS (pH7.0) was added to each well. Next, after adding 50 μL of original viral solution, they underwent two folds serial dilutions. Finally, 50 μL of 0.5% turkey red blood cells were added and mixed. The mixture was then incubated for approximately 30 minutes at room temperature. The number of positive reactions showing agglutination was observed and recorded to calculate virus titer.
To prepare the nasal swab specimens, a sterile swab was carefully inserted into the nostril with the most secretions under visual inspection. Using a gentle rotation, the swab was pushed until a weak resistance occurred at the level of the turbinate (less than one inch into the nostril). Next, the swab was rotated a few times against the nasal wall. To test the RDT kit, a test strip was inserted into the tube containing the 300 μL of extraction solution and allowed to sit at room temperature prior to testing. After preparing the nasal swab, the sample was inserted into the tube and swirled at least six times while pressing the head against the bottom and side of the tube (the swab head should be rolled and squeezed against the inside of the tube as it is removed). Finally, the test strip was inserted into the tube containing the sample-extracted solution. The results were interpreted after 10 minutes. Some positive results appeared sooner, but results were not read after 30 minutes.
For confirmation of cultured influenza A/H1N1, the viral RNA was isolated from the infected MDCK cells. RT-PCR was performed with the commercialized Influenza A(H1N1) Detection Kit by using primers targeting the novel influenza (GeNet Bio, Nonsan, Republic of Korea). RNA was directly extracted from the specimens or virus cultured-supernatant using the QIAGEN® Viral RNA mini kit (Qiagen, Hilden, Germany). cDNA synthesis was accomplished at 42°C for 30 minutes. Next, the DNA was amplified by 40 cycles of PCR with three steps: denaturation at 94°C for 20 seconds, annealing at 54°C for 20 seconds, and elongation at 72°C for 30 seconds. Finally, an additional elongation step (72°C/5 min) was carried out. The amplified gene products were analyzed with 2% agarose gel electrophoresis. The size of the amplified target was 170 base pair for novel influenza A/H1N1 and 350 base pair for seasonal influenza A/H1N1.