1 平成24年度組織的な若手研究者等海外派遣プログラム帰国報告書

平成２４年度組織的な若手研究者等海外派遣プログラム
帰国報告書（研究留学）
提出日：平成 25 年 2 月 27 日
フリガナ
氏
名
派遣先名
（国名）
カザマ
シノブ
風間しのぶ
所属
大学院人間文化創成科学研究科リサーチフェロー
スイス連邦工科大学ローザンヌ校（スイス）
(日本出発日 )
派遣期間
研究テーマ
（日本到着日）
平成 24 年 2 月 26 日
～
平成 25 年 2 月 15 日
病原ウイルスの不活化機構の解明と病原リスク評価基準の検討
①
ローザンヌでの生活と研究についての総括
派遣先のスイス連邦工科大学ローザンヌ校（EPFL）はスイスのレマン湖北岸のローザンヌという都市にある。
まず、ローザンヌに到着した時、その美しい風景に感動した。レマン湖沿岸を走る鉄道からの景色、ローザン
ヌは坂が多いため、レマン湖岸からだけでなく、高台からの景色、特に対岸に見えるフランスのエビアン等の
町の夜景はとても美しく何度見ても飽きなかった。
ローザンヌの公用語はフランス語であるが、スイスは地域によって、スイスドイツ語、フランス語、イタリ
ア語またはロマンシュ語が公用語とされている。多くのスイス人は複数言語を話すことができ、ローザンヌの
町でも多くの人が英語も堪能であったため、フランス語の話せない私でも、日々の生活において大きな問題は
なかった。
そんなローザンヌでの生活にも初めはいくつか苦労した点があった。スイスの物価が高いということは広く
知られているが、それ以外に、まずは、住居である。物件も少なく、スイス人であっても、部屋がなかなか見
つからないということで、私も大変苦労した。
次に洗濯で、洗濯機と乾燥機をアパート住人で共有し、各家庭に使用可能な曜日と時間帯が割り当てられる
ことが多いため、2 週間に一度しか順番が回ってこないという方の話も聞いた。
スーパーや商店等の閉店時間が早く、日曜祝日は休みであるため、日々の買い物に苦労した。日本での生活
と異なり、初めは不便と感じていたが、徐々に慣れていった。
日曜祝日は町では何もすることがないが、夏はハイキングや湖、冬は（残念ながらスキーには行けなかった
が）そりを楽しむことができた。スイスの自然は本当に素晴らしく、日本にいる時には全く興味がなかったが、
晴れた日には必ずと言っていいほどハイキングに行った。
EPFL は多くの留学生がいるが、私の受け入れ先の研究室は、ほとんどがスイス人であった。皆まじめで優
秀な学生ばかりだった。また、皆スポーツ好きで、昼休みや仕事の後、よく体を動かしに出かけた。
日本での日常とは異なるものであったが、次第に慣れ、
また、それが私の日常となり、日々の研究も効率的に進め
られたと思う。
研究生活も終盤に差し掛かった時点で、最後にもう少し
時間があれば、とも思ったが、約一年の留学生活で、本当
に素晴らしい仲間と出会い、多くの技術を得ることができ
た。今回の留学で得たものは数えきれないほどあるが、こ
の留学の機会を与えてくださった皆様、支えてくださった
皆様に心から感謝し、この気持ちを忘れずに、今後の研究
スイスでの休日
に生かしていきたい。
1
②
氏名：風間しのぶ
Research report
Introduction
The introduction of sewage systems has improved
public health in cities worldwide. However, 90% of the
world’s wastewater is not properly treated, and this
wastewater continues to pollute the water’s water
resources (WHO, 2002). Because many deaths are
caused by water contaminated with pathogens (WHO,
2002), the improvement of sanitation in developing
countries is of critical importance. Recently, dry toilets is
available
for
improved
sanitation
in
developing
countries; these toilets are inexpensive and do not
require development of costly infrastructure. In addition,
because dry toilets can recycle human waste as fertilizer, these systems have many advantages from the
viewpoint of preserving water resources and promoting nutrient recycling (Winblad et al., 2004). However,
care is required in the safe handling of human waste, both for reuse and for disposal.
Ammonium is naturally present in urine and anaerobic digested sludge. In its uncharged dissolved form
(NH3(aq)), ammonium has been shown to have biocidal effects (Cramer et al., 1983), and can therefore act
as an in-situ disinfectant. However, the effect of ammonia on enteric viruses, which are commonly found in
human excreta, remains unclear.
Objectives
The goal of this study was to characterize the virucidal effects of ammonia on different viruses and in
different physical–chemical conditions, and to identify the mechanisms of viral inactivation. We also explored
the contribution of NH3(aq) to virus inactivation in natural solutions, such as in those stored in urine.
Materials and Methods
To investigate the effects of aqueous ammonia on the inactivation of viruses, we conducted tests using
several types of viruses in a range of environmental conditions. Inactivation of coliphage MS2 (ssRNA) in
ammonium carbonate buffer (AmCa) was extensively studied over a range of environmentally relevant
NH3(aq) activities, temperatures, pH values, and ionic strength conditions. These results were compared with
patterns of inactivation of other viruses with different capsid compositions (e.g., coliphages GA and fr
(ssRNA)) and different genome types (coliphage ΦX174 (ssDNA), as well as results using human adenovirus
(dsDNA), mammalian reovirus (dsRNA)) and echovirus (ssRNA). Inactivation in ammonium carbonate buffer
was then compared with inactivation in stored urine solutions.
Finally, a detailed evaluation of the effects of NH3(aq) on the different virus components (genome or capsid)
was conducted using quantitative reverse transcription polymerase chain reaction (RT-qPCR) and
matrix-assisted laser desorption/ionisation- time of flight (MALDI-TOF) mass spectrometry (Wigginton et al.,
2012). In addition, the loss of vital virus functions (binding and genome infectivity) was assessed by binding
assays and injection assays (Wigginton et al., 2012) by using MS2.
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氏名：風間しのぶ
Results and discussion
Figures1a shows that MS2 inactivation increases with increasing active fractions of NH3(aq), in turn, is
influenced by pH, ionic strength and temperature. The insets in Fig. 1a and b show that, of all the
physical–chemical parameters, high temperatures and pH values > 9 appeare to synergistically enhance the
virucidal effect of NH3(aq) beyond causing a simple increase in its active fraction.
An additional inactivation effect was observed in stored urine as compared with in ammonium carbonate
buffer, as shown in Fig. 2. It has been suggested that additional inactivating agents, other than NH3(aq), are
present in urine.
Figure 3 shows the inactivation rate constants of viruses under various conditions. The DNA viruses generally
show higher resistance to NH3(aq) than do RNA viruses. It has been suggested that genomes might be the
main degradation target. However, differences in inactivation rates were also observed among RNA viruses.
The echo virus was inactivated especially quickly (data not shown). This suggests that additional mechanisms
other than genome degradation are involved in inactivation by NH3(aq).
We also applied RT-qPCR to MS2 samples treated with AmCa to test for infectivity. In addition, the
degradation of the MS2 genome in AmCa was studied using naked MS2 genome (extracted RNA). However,
degradation of the MS2 genome could not be distinguished by RT-qPCR.
Fig.1a Inactivation effect to MS2 by NH3(aq)
Fig.1b Inactivation effect to MS2 by pH
activity
35 ˚С
20˚С
Fig.2 Inactivation rate constants of MS2 in
AmCa (blue) or urine with or without
dilution (yellow).
Fig.3 Inactivation rate constants of viruses.
The ratios in this figure show urine:water.
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氏名：風間しのぶ
35˚С, pH9
---------------Binding------------
Infectivity
----Run1
Run2
Run3
Fig.4 Rate constants of MS2 binding ability
Capsid protein peptides*
loss and inactivation rate constant of
Fig.5 Ratios of protein loss to log loss infectivity
*1-7,ASFNTQF;8-25,VLVDNGGTGVDTVAPSNF;26
-32,ANGVAEW;
33-42,ISSNSRSQAY;39-43,SQAYK;44-49,VTCSVR
;50-56,QSSAQNR;59-82,TIKVEVPKVATQTVGGVE
LPVAAW;67-82,VATQTVGGVELPVAAW;84-106,S
YLNMELTIPIFATNSDCELIVK;107-113,AMQGLLK;
113-129,DGNPIPSAIAANSGIY.
MS2 (infectivity).
Blue bars, in VDB (control); Red bars,
in AmCa.
40mM
Next, we investigated MS2 capsid protein degradation by using binding assay and MALDI-TOF mass
spectrometry. We detected no difference in the rate constant of MS2 binding ability using phosphate buffer
(VDB) as a control as compared with that using AmCa (Fig.4); however, MS2 lost infectivity, indicating that
there was no loss of binding ability due to NH3 (aq). As comparing with the effect of VDB controls and
phosphate carbonate buffer (PCa; without NH3), no additional capsid protein damage by NH3(aq) was observed
(Fig. 5). Therefore, the results in Figs.4 and 5 indicate that NH3(aq) decreases genome replication ability.
Conclusions
Under environmentally relevant conditions, inactivation by ammonium buffered solutions is solely
characterized by NH3(aq) activity and temperature. In urine, additional unknown inactivation compounds may
accelerate virus loss.
Viruses showed heterogeneous behavior towards NH3(aq) inactivation, mainly according to their genome
type, and to a lesser extent according to their capsid composition. Notably, our results suggest that only ssRNA
viruses are significantly inactivated by NH3(aq). Therefore, we should take care to assess inactivation effects
on viruses with different types of genomes.
We consider that viruses are mainly affected by NH3(aq) through damage to genome replication functions.
However, the mechanisms of inactivation remain unclear and require further investigation.
References
WHO (2002) Reducing risks, Promoting healthy life. World health report 2002, World Health Organization,
Geneva.
Winblad U., Hébert M.S., Calvert P., Morgan P., Rosemarin A., Sawyer R. and Xiao J. (2004). Ecological
Sanitation revised and enlarged edition. Stockholm Environment Institute, Stockholm.
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氏名：
風間しのぶ
W. N. Cramer et al. (1983) Kinetics of virus inactivation by ammonia, Applied Environmental Microbioogy., 45,
760–765.
K. Wigginton et al. (2012) Virus inactivation mechanisms: impact of disinfectants on virus function and
structural integrity, Environmental Science Technology,46 (21), 12069–12078
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