22E08
Analysis of the inactivation mechanism of bacteriophage MS2 and φX174
by atmospheric pressure non-equilibrium plasma
大気圧非平衡プラズマによるバクテリオファージ MS2 および φX174 の不活化メカニズムの解析
Yoshimasa Tanaka, Hachiro Yasuda, Hirofumi Kurita, Kazunori Takashima, Akira Mizuno
田中良昌, 安田八郎, 栗田弘史, 高島和則，水野彰
Toyohashi University of Technology
1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
豊橋技術科学大学 〒441-8580 愛知県豊橋市天伯町雲雀ヶ丘 1-1
Atmospheric pressure non-thermal plasma has attracted attention as a method of virus inactivation
because it can efficiently inactivate various viruses in a short time and at low temperature. However, thepredominant factor in the inactivation is not clear yet. Bacteriophages, consisting of nucleic acid packed
into proteins, are suitable for the study of the inactivation mechanism due to their simple structure.
Analyzing the inactivation of bacteriophage MS2 and φX174 should be helpful in understanding theinactivation of influenza virus by plasma. When these bacteriophages are inactivated by dielectric barrierdischarge (DBD), the damage should exist either on viral nucleic acids, coat proteins or both. It is possible
to extract the nucleic acids from the bacteriophage so that the damaged coat proteins can be removed toavoid the effect of the proteins. And, the nucleic acids extracted from plasma-inactivated phage can beassayed its plaque forming activity by transfection of the nucleic acids. Using this assay, the damages ofviral nucleic acids and coat proteins have been determined separately. As a result, the main cause ofinactivation of bacteriophage MS2 and φX174 by DBD exposure should be due to the damage of MS2
RNA and φX174 coat proteins, respectively.

1．Introduction
Inactivation technology of viruses have been
actively studied, because there is a serious problem
which is communicable viral infection caused byinfluenza virus. In recent years, atmosphericpressure non- thermal plasma has attracted attention
as a method of virus inactivation because it canstrongly inactivate in a short time and at low
temperature [1]. However, the predominant factor
in the inactivation is not clear yet. Bacteriophages,
consisting of a nucleic acid packed into proteins,are suitable for the study of the inactivationmechanism due to their simple structure [2]. In thisstudy, bacteriophage φX174 which is consisted ofonly viral DNA and protein was investigated using
atmospheric pressure dielectric barrier discharge(DBD) known as having highly microbicidal effect[3]. When bacteriophage is inactivated by DBD, thedamage should exist either on viral nucleic acids,
coat proteins or both. Bacteriophage φX174 andMS2 have some characteristics similar to influenza
virus. Analyzing the inactivation of bacteriophage
φX174 should be also helpful for understandinginactivation of influenza virus.

2．Experimental part
Figure 1 shows a DBD generator used in this study.
A high voltage AC power supply was used togenerate DBD in air gap (3 mm). In this report,
discharge experiments were performed under fixed
electrical conditions except the exposure time. The
effective voltage, the frequency of the applied
voltage, and the input power were 14 kV, 2.00 kHz,and 8.8 W, respectively.
A 50 μL aliquot of φX174 phages sample solution
was spotted and widely spread to 3-4 cm2 on a PET
film and the DBD was immediately applied for theintended time. After that the sample solution wasrecovered by additional 50μL of the distilled wateron the surface of the PET film. For themeasurement of phage activity, the recovered phagesolution was mixed with Escherichia coli
ATCC13706 and poured on an agar media plate.
Phage plaques on the plates were counted afterincubation at 37℃ for 24 hrs. On the other handthe activity of DNA extracted from DBD-treated
φX174 phages were measured by transfection,
which is a method to form infectious φX174 phage
particle by mixing purified DNA with competentAC High voltage
power supply
Acryl board
Electrode(Stainless mesh)Φ50 mmDischarge conditions
Applied effective voltage : 14 kV
Frequency: 2.00 kHz: 8.8 W
Discharge powerDielectrode(Teflon sheet)3 mm

wet sample
50 μL

Alminum plate
PET film

100

Relative PFU

E.coli cells. Transfection allows measurement ofDNA damage only without being affected by thedamages of coat proteins. Damages of coat proteins
were analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

10-2
10-4

10-6
10-8

Phage activity
DNA activity

10-1010-120102030
Treatment time (sec)Fig. 2 Inactivation profiles of DBD-treated φX174
phage and φX174 DNA (PFU : plaque forming unit)
M

0Treatment time (sec)10203040M

Fig. 1 Atmospheric pressure DBD generator

3．Results and discussion
Figure 2 shows the inactivation profile of theφX174 phage after DBD treatment, as well as theactivity of DNA extracted from DBD-treatedphages obtained from the transfection. These
profiles were normalized with each control sample(0 sec samples) to give the PFU (plaque formingunit) value of 100. The profile exhibited a decreasein activity of φX174 phage. The activity of DNA
also decreased in the first 10 sec. However, theywere retained at long time from 20 sec to 40 sec.
These results show that the predominant factor inthe inactivation of φX174 phage is not DNAdamage.
Figure 3 shows the analysis of coat proteins fromDBD-treated φX174 phage by SDS-PAGE. Thesecoat proteins degraded with the treatment time. In
addition, band shifts with the treatment time
showed that some modifications of proteins exist.
These results show that despite φX174 phageactivity decrease with treatment time, the activity ofDNA extracted from DBD-treated phage wasretained. Moreover damages of coat proteinsobtained from DBD-treated φX174 phage exist.
Therefore the cause of inactivation of φX174 phage
by DBD exposure should be mainly due to thedamages of coat proteins.
In the case of MS2 phage, the activity of phageand viral RNA similarly decreased with DBD
treatment time. However, coat proteins did not sodegrade. Consequently, the main cause ofinactivation of MS2 phage by DBD exposure
should be due to the damages of RNA.

40

φX174 phagecoat protein

M : Kaleidoscope standard

Fig. 3 Analysis of the DBD-treated φX174 phagecoat protein by SDS-PAGE

4．Conclusions
Using bacteriophage φX174 and MS2, we havedeveloped an assay method based on extraction of
the viral nucleic acids from plasma-treatedbacteriophage and transfection of the nucleic acids
to the host bacteria. This assay was applied to thebacteriophage φX174 and MS2 which wereinactivated by the treatment of atmosphericpressure non-thermal plasma. As a result, despite
φX174 phage activity decrease with treatment time,
the activity of φX174 DNA extracted from DBDtreated phages was retained. And, the activity ofMS2 phage and MS2 RNA similarly decreased withDBD treatment time. MS2 coat proteins did notdegrade so much. These results exhibited the maincause of inactivation of bacteriophage φX174 andMS2 by DBD exposure should be due to thedamage of φX174 coat proteins and MS2 RNA,respectively.
References[1] M. Laroussi, Plasma Processes and Polymers 2:
391, 2005.
[2] H. Yasuda, T. Miura, H. Kurita, K. Takashima andA. Mizuno, Plasma Processes and Polymers 7:
301-308, 2010.
[3] M. Tanino, W. Xilu, K. Takashima, S. Katsura, A.
Mizuno, International Journal of Plasma
Environmental Science & Technology 1: 102-107,
2007.