Fig 1.
Viral titration assays demonstrate that nitrogen gas plasma treatment of adenovirus results in a decrease in viral titer.
A cell culture medium of adenovirus vector (AxCAwt2)-infected HEK293 cells (1.0 x 109 PFU/ml) was air-dried onto a glass coverslip and treated with nitrogen gas plasma using a BLP-TES device (NGK Insulators, Ltd., Nagoya, Japan) at 0.5 atmospheric pressure. The recovered samples were then subjected to a viral titration assay using HEK293 cells as described in Materials and Methods. The number of plaque forming units (PFU) per ml of adenovirus treated with nitrogen gas plasma at 1.5 kpps for 0, 5, or 15 min (N = 4) was then determined. The 0 min samples are recovered from coverslips subjected to 0.5 atmospheric pressure without plasma treatment. Differences where p<0.05(*) versus control (0 min) were considered significant. No plaques were detected after treatment for 15 min.
Fig 2.
5-bromo-3-indolyl-β-D-galactopyranoside staining detected a decrease in viral titer of adenovirus after nitrogen gas plasma treatment.
A cell culture medium of adenovirus vector (AxCAiLacZ)-infected HEK293 cells (1.0 x 109 PFU/ml) was air-dried onto a glass coverslip and treated with nitrogen gas plasma using the BLP-TES device at 1.5 kpps for 0, 5, and 15 min. After the treatment, adenovirus was recovered from the spots and subjected to 5-bromo-3-indolyl-β-D-galactopyranoside staining as described in Materials and Methods.
Fig 3.
Infectious titer assay using antibody against adenovirus hexon protein show decreased viral infectivity of adenovirus after nitrogen gas plasma treatment.
A cell culture medium of adenovirus vector (AxCAwt2)-infected HEK293 cells (1.0 x 109 PFU/ml) was air-dried onto a glass coverslip and treated with nitrogen gas plasma using a BLP-TES device at 1.5 kpps for 0, 5, and 15 min. After treatment, adenovirus was recovered from the spots and then subjected to an infectious titer assay using antibody against adenovirus hexon protein to determine the number of infected cells.
Fig 4.
Nitrogen gas plasma treatment induces damage to the genomic DNA of adenovirus.
A cell culture medium of adenovirus vector (AxCAiLacZ)-infected HEK293 cells (1.0 x 109 PFU/ml) was air-dried onto a coverslip and treated with nitrogen gas plasma using the BLP-TES device at 1.5 kpps for 0, 5 and 15 min. The recovered samples were then subjected to polymerase chain reaction (PCR) analysis using primers designed to amplify the hexon gene of adenovirus. A PCR product of the anticipated size was observed at 0 min (indicated by arrowhead), but the intensity of the band progressively diminished after gas plasma treatment for 5 and 15 min. The molecular marker lane (M) is 100 kb DNA ladder. Numbers shown on the left correspond to the molecular size (bp).
Fig 5.
The level of intact viral genomic DNA decreased following nitrogen gas plasma treatment.
A real-time PCR assay was performed using cell culture medium of HEK293 cells infected with AxCAiLacZ vector (1.0 x 109 PFU/ml) after treatment with nitrogen gas plasma for 0, 5, and 15 min (N = 4). The primers were designed against the adenovirus hexon gene. The amount of intact adenovirus genomic DNA decreased after nitrogen gas plasma treatment. The average titer of 0 min samples was taken as 100%. Differences where p<0.01(**) versus control (0 min) were considered significant or not significant (NS).
Fig 6.
Nitrogen gas plasma treatment has no effect on adenovirus hexon protein.
(A) Nitrogen gas plasma-treated cell culture medium of HEK293 cells infected with adenovirus vector (AxCAiLacZ) (1.0 x 109 PFU/ml) was subjected to immunochromatography using the Quick Navi-Adeno kit (Denka Seiken Co., Ltd., Tokyo, Japan). The bands corresponding to adenovirus hexon protein are indicated by an arrowhead. C: Control line; T: Test line. (B) The band intensities of test lines of the immunochromatography in three independent experiments were compared using Image J software. The band intensity of the test line for the 0 min sample in each experiment was taken as 100%. Band intensities of test lines of adenovirus samples treated for 0, 5 and 15 min are shown. Immunochromatography showed no significant change in the hexon protein of adenovirus after nitrogen gas plasma treatment for up to 15 min.
Fig 7.
No difference in the adenovirus hexon and penton proteins was detected after nitrogen gas plasma treatment.
(A) Cell culture medium of adenovirus vector (AxCAiLacZ)-infected HEK293 cells (1.0 x 109 PFU/ml) was treated with nitrogen gas plasma for 0, 5 or 15 min (BLP-TES, 1.5 kpps). The treated adenovirus samples were then subjected to Western blotting with a polyclonal anti-adenovirus antibody. (B) The band intensities from three independent Western blots were compared using Image J software. The band intensity for 0 min in each blot was taken as 100%. Band intensities of adenovirus samples treated for 0, 5 and 15 min are shown. No significant change was detected in the hexon (116 kDa) and penton (80 kDa) capsid proteins of adenovirus after nitrogen gas plasma treatment for up to 15 min.
Fig 8.
Reactive chemical species are generated during operation of the nitrogen gas plasma instrument.
Chemical indicator strips were used to detect the generation of reactive chemical species during operation of the nitrogen gas plasma device. Specifically, we tested for hydrogen peroxide [Merckoquant® Peroxide Test 0.5–25 mg/l H2O2, Merck KGaA, Darmstadt, Germany] (A), nitrate (NO3-) [Merckoquant® Nitrate Test 10–500 mg/l NO3-, Merck KGaA] (B), and nitrite (NO2-) [Merckoquant® Nitrite Test 2–80 mg/l NO2-, Merck KGaA] (C). In each case, the strip was placed on the earth electrode of the BLP-TES instrument. Nitrogen gas plasma treatment was then performed at 1.5 kpps for 0, 5, or 15 min (N = 3). After treatment, the strips were immediately dipped into pure water (Otsuka distilled water; Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan). The concentration of hydrogen peroxide, nitrate, and nitrite was estimated from the change in colour of the corresponding chemical indicator strip, which was scanned and compared with a calibration curve of references. Differences where p<0.05(*) and p<0.01(**) versus control (0 min) were considered significant or not significant (NS).