Abstract(本文)

Water contaminated with pathogen has been a significant cause of human decease around the world (WHO 2002). Water treatment and disinfection has been essential and required to mitigate the occurrence of waterborne pathogen. Among the disinfection methods, chlorine, ozone, and ultraviolet (UV) light are typical disinfectants that are currently used in the wastewater and water purification plants. The disinfection can inactivate microorganisms in various ways. The disinfection mechanisms and their effects on microorganism are depended on the types of disinfectants and microorganisms. This research was carried out for evaluating the effect of chlorine, ozone, and UV light on viral nucleic acid and capsid integrity by PCR based techniques. Cell culture, ethidium monoazide treatment coupled with real-time polymerase chain reaction (EMA-PCR), and real-time polymerase chain reaction (PCR) were used to detect the infectivity, damage on viral genome, and damage on viral nucleic acid that inactivated or destroyed by disinfectants. The different kinds of microorganisms were investigated that were poliovirus,

The structure of this dissertation is: the background and literature reviews were in Chapter 1 and 2. These chapters described the research objective and strategy, the background from the previous researches, as well as the importance and requirement of our research. Chapter 3 described the analytical methods that were applied in our study. The preliminary investigations and some development of the methods, which were applied in the inactivation studies of viruses, were described in Chapter 4 and 5. The Chapter 6, 7, and 8 related to the study of disinfection mechanisms of viruses by chlorine, ozone, and UV light, respectively. Finally, Chapter 9 described the overall conclusions and recommendations from this study. The obtained results from each chapter were explained below.

Chapter 4 was separated into two main parts. The first part related to the establishment of cell culture for adenovirus that would be applied to study the inactivation adenovirus in the Chapter 7 and 8. The Hep-2 cell and A549 cell line were cultivated and used as a host cell for adenovirus. The adenovirus 2, 3, 5, 7, 40, and 41 types were tested with those two host cells. The results showed that adenovirus 5 was able to propagate in these Hep-2 and A549 cell while other types of adenoviruses were not. The plaque could not be observed when adenovirus 5 was tested with Hep-2 cell line. However, we could observe plaque clearly when A549 was used as a host cell for adenovirus 5. The plaque assay for adenovirus 5 was successfully established and would be applied in other Chapters. The second parts of this Chapter focused on the investigation of the effect of ethidium monoazide (EMA) treatment on bacteriophage Qβ, MS2, and T4, poliovirus 1 (PV1), murine norovirus (MNV), and adenovirus 5 (Ad5). This investigation was done before the EMA treatment was applied in Chapter 6, 7, and 8. The EMA could penetrate to intact bacteriophage Qβ and MS2 and inactivate these bacteriophages at the EMA concentration of 5, 10, and 50 μg/mL. So it was not applicable to apply EMA treatment with two types of bacteriophages. The EMA did not have an effect on intact poliovirus 1, murine norovirus when the EMA treatment at the concentration of 5, 10, and 50 μg/mL was applied. In the case of adenovirus 5, the EMA treatment did not showed the effect on the intact adenovirus 5 at the concentration of 5 and 10 μg/mL but it inactivated adenovirus 5 at the concentration 50 μg/mL. When the EMA-PCR and PCR were used to measure the concentration of PV1, MNV, and Ad5, the results from these two assays was quite similar. EMA treatment did not affect the PCR analysis, which consisted of RNA/DNA extraction, reverse transcription, and polymerase chain reaction. In addition, results implied that our virus stocks consisted of integrity virus and these viruses could protect viral genome from EMA treatment. The EMA treatment at the concentration of 50 μg/mL would be applied with PV1 and MNV, at the concentration of 10 μg/mL would be applied with Ad5 in the following Chapters.

In Chapter 5, the continuous quench flow system was developed for study the rapid ozone disinfection kinetics in Chapter 8. A CQF reactor was established for studying the viral inactivation. A two-channel peristaltic pump was used to drive both ozone and viral solution into reaction chamber (mixer), which was made from T-connecter (inlet diameter 1 mm; inside diameter 4 mm; outlet diameter 2 mm) with magnetic stirrer placed inside. The reacting mixture then flowed through Teflon tube for prolonging contact time before the mixture flowed to collection tubes containing quenching solution. The samples were separately collected for measuring residual ozone concentration and microbes. The contact time between two substances was controlled by adjusting the flow rate of pump and the length of the Teflon tube. The complete mixing between two solutions was ensured by observing changed color of base and acid indicators. The mixing ratio between two solutions was verified by investigating the sodium chloride concentration reducing to half of initial concentration. Before this established reactor was applied to study the viral inactivation by ozone, this CQF reactor was applied to study the viral inactivation by chlorine first. In order to verify the consistence of CQF reactor to measure the inactivation at very short contact time, the disinfection kinetics of bacteriophage Qβ after chlorination was investigated by both CQF and batch reactor. The inactivation of bacteriophage in CQF reactor was investigated from contact time varying from 0.7 second to 10.2 seconds and this inactivation was compared with inactivation results from batch reactor at longer contact time. The results showed that the inactivation rate fitted to linear regression between these two reactors was not significant difference based on the statistical analysis (p > 0.05). These results implied that the inactivation results from CQF reactor were consistent with inactivation results from batch reactor. The inactivation data at short contact time is warranted.

Chapter 6 related the usefulness of EMA-PCR and PCR for the evaluation of damage on viral genome and capsid by chlorine. The viral infectivity, damage on viral genome, and damage on viral nucleic acid were evaluated by Cell culture, ethidium monoazide treatment coupled with real-time polymerase chain reaction (EMA-PCR), and real-time polymerase chain reaction (PCR). The chlorine disinfection at different concentration of 0.1, 0.25, 0.5, and 1 mg/L was applied with PV1 and at concentration of 0.25, 0.5 and 1 mg/L was applied with MNV. After PV1 and MNV were inactivated by chlorine, the inactivation rate results obtained from EMA-PCR was higher than to the results obtained by PCR at the initial chlorine concentration of 0.25, 0.5, and 1 mg/L for PV1 and 0.5 and 1 mg/L for MNV, respectively. At this rate, the disinfection mechanism of chlorine was mainly on the damage of viral capsid. The viral inactivation rate detected by plaque assay was lower than the reduction detected by EMA-PCR. Chlorine might react with viral particle and lead to the loss of viral infectivity but it did not cause enough damage on viral capsid, which let EMA penetrate to viral capsid. For instance, the damage might cause at the viral attachment site. At the clorine concentration 0.1 mg/L for PV1 and 0.25 for MNV, the reduction of viruses was not observed by EMA-PCR and PCR at short contact time. Chlorine might not cause severe damage on viral capsid. The capsid still protected the viral genome from EMA. However, the loss of viral infectivity might come from the destruction of antigenicity of virusese and viruses unable to attach to the host cell.

The EMA-PCR and PCR were used for evaluating the effect of ozone on viral nucleic acid and capsid integrity in Chapter 7. In order to investigate the rapd inactivation by ozone, a continuous quench-flow (CQF) reactor, which was established in Chapter 5, were applied to study the disinfection kinetic of microbes by ozone at very short contact time e.g. 0.7 second in this chapter. By applying CQF reactor, the positive results of remaining virus after ozonation could be obtained. The CQF reactor was appropriate for studying the fast inactivation rate of microbes by both chlorine and ozone at the contact time as short as 0.70 second. By using the CQF reactor to study the inactivation of viruses at short contact time, we could elucidate the inactivation of viruses clearer than what we learned from batch reactor. Ozone had an ability to cause damage to viral capsid and genome of PV1. The destruction by ozone seemed to primary on viral capsid as we could observe from the higher reduction rate of viruses from EMA-PCR than PCR.

Chapter 8 described the investigation of damage on viral capsid by low-pressure UV lamp (LP UV lamp) and medium-pressure UV lamp (MP UV lamp). When polioviruses and adenoviruses were inactivated by LP UV lamp, the inactivation results detected by PCR and EMA-PCR showed no difference even at high UV fluence of 1000 mJ/cm2. However, the results of EMA-PCR showed higher inactivation rate than the results of qPCR when the polioviruses and adenoviruses were inactivated by MP UV, just at UV fluence above 300 mJ/cm2. Additionally, the difference became larger as the UV fluence was increased. At the UV fluence of 500 mJ/cm2, the difference was more than 0.4-log and 1-log for the poliovirus and adenovirus, respectively. The difference of results between PCR and EMA-PCR implied that EMA could penetrate to inactivated damaged viral capsid, which had complete viral nucleotide, and bind to viral DNA/RNA. Therefore, the difference between inactivation rate of viruses after MP UV lamp observed by EMA-PCR and PCR meant that MP UV lamp has a potential to cause the damage on viral capsid. The MP UV lamp emitted broad wavelength spectrum from 200 to 600 nm while LP UV lamp mainly emitted UV light at a wavelength of 254 nm. Therefore, the effectiveness of UV wavelength on viral capsid was investigated. The difference between PCR and EMA-PCR at the UV wavelength around 230 to 240 nm is larger than others. The reasons might be that the composite of viral capsid is protein. This data agreed with the UV absorbance profile of proteins and amino acids, which showed high absorbance at low UV wavelength. The previous study found that MP UV lamp enhanced the inactivation of adenovirus over LP UV lamp. Based on our study, MP UV possibly caused damage on viral capsid, which may enhance the inactivation of adenovirus. This finding contributes to better understanding in UV disinfection mechanism and leads to the improvement of UV disinfection in the future.

In this study, the EMA-PCR and PCR had a merit to investigate the damage on viral genome and capsid of viruses. The understanding of disinfection mechanisms from this study has a benefit to provide an effective disinfection method to control viruses. In addition, it could use as an information for develop PCR-based method to selectively detect infectious viruses in the future.

本論文は、Application of PCR-based techniques for evaluating the effect of chlorine, ozone and ultraviolet light on viral nucleic acid and capsidintegrity (塩素、オゾンおよび紫外線がウイルス核酸およびカプシドに及ぼす影響のPCRに基づく手法による評価)と題する。水野微生物学的安全性の確保のために、ノロウイルスをはじめとするヒト腸管系ウイルスの消毒手法およびその有効性の確認が重要な課題とされており、分子生物学的手法を含めて研究したものである。本論文は以下の9章で構成されている。

第1章では、水中の病原ウイルスの消毒の必要性と様々な消毒法、および消毒後のウイルス測定法を概観し、ウイルス消毒を研究することの意義ならびに本論文の目的と構成を示している。

第2章では、水中ウイルスについての既存の知見およびその処理法に関する既存の知見をまとめている。現在問題となっている水中のウイルスおよび測定法について整理し、塩素消毒、紫外線消毒ならびにオゾン処理における微生物の不活化に関する先行研究をまとめ、既存の知見を整理している。

第3章では、ウイルスの培養法および分子生物学的手法によるウイルス検出法など、本研究で用いた実験方法を記述している。

第4章では、前半部において、アデノウイルスの培養法を確立している。Hep-2細胞とA549細胞を培養してアデノウイルスの宿主として用い、アデノウイルス5型の培養に成功し、A549細胞においてはプラックの観察にも成功したことから、アデノウイルスの感染価を測定する方法が確立し、消毒実験の効果の測定に用いることが可能となった。後半部では、エチジウムモノアザイド(EMA)処理による効果を調べている。対象としたウイルスは、大腸菌ファージQβ、MS2、ポリオウイルス1型、マウスノロウイルスおよびアデノウイルス5型である。感染価を有する大腸菌ファージQβおよびMS2に対して、EMAはカプシドをすりぬけてRNAと反応したが、ポリオウイルス1型、マウスノロウイルスおよびアデノウイルス5型に対してはEMAはRNA/DNAと反応しないことを確認した。その結果、QβおよびMS2に対してはEMA処理を用いた処理は適さないが、ポリオウイルス、マウスノロウイルス、アデノウイルスに対してはEMA処理はカプシドの完全性を調べるための手法として有効であることを明らかにした。なお、EMA処理に用いるEMA濃度は、ポリオウイルス、マウスノロウイルスに対しては50μg/ml、アデノウイルスに対しては10μg/mlが適していることを確認した。

第5章では、オゾンによる短時間の不活化効果を測定するための装置として、連続反応装置(Continuous Quench Flow, CQF)の開発を行っている。この装置は第8章で用いている。本装置は、ぺリスタポンプによりオゾン溶液と試料を等量輸送して混ぜる装置であり、流入口の内径1mm、流出口内径2mm、混合部サイズ4mmのT型のコネクタを用い、内部に微小スターラーを入れている。混合部より以降にチューブを備えることにより、液の混合後の反応時間をコントロールする装置である。本装置は、混合部における完全な混合が重要であるため、酸性溶液とアルカリ性溶液にpH指示薬を用いることにより、混合状態を視認して混合状態を確認した。また、混合比率が等しいことも塩化ナトリウム溶液と性盛衰を用いた実験により確認している。また、塩素とQβを用いて0.7秒から10秒までの接触時間による消毒実験を行い、より長い接触時間のバッチ式実験と違いがないことを確認している。これらの結果より、CQF反応装置は設計通りの実験が可能であり、かつ、0.7秒という非常に短時間の消毒実験を行うことが可能となった。

第6章では、塩素消毒により生じたウイルスへの損傷の測定におけるEMA-RT-PCRの有用性について論じている。ウイルスの感染価、ウイルスカプシドの損傷、並びにウイルス核酸の損傷について、細胞培養法、EMA- RT-PCR法、ならびにRT-PCR法により調べている。塩素消毒の初期濃度は0.1、0.25.0.5及び1mg/Lとし、ポリオウイルスおよびマウスノロウイルスの不活化効果を調べている。EMA- RT-PCR法では、RT-PCR法よりも不活化速度が大きいという観察結果が得られた。低濃度の塩素消毒ではこの差があまり見られず、EMAはカプシドを通過できない状態でもウイルスが不活化しているという現象が見られた。初期塩素濃度0.5mg/L以上では、塩素はウイルスのカプシドに損傷を与えている可能性が高いことが明らかとなった。

第7章では、オゾンによるウイルスの不活化の状況を調べるため、CQFリアクターを用いて実験している。オゾンによるウイルスの不活化は非常に短時間で生じるので、0.7秒の接触時間をはじめ、経時的なウイルスの定量値を、プラック法、EMA-RT-PCR法およびRT-PCR法を用いてしらべ、オゾンによるポリオウイルスの不活化、カプシドの損傷および核酸への損傷を調べている。CQF反応装置を用いることにより、バッチ型消毒試験によるよりも詳細にウイルスの不活化を調べることができた。EMA-RT-PCRによりRT-PCRよりも不活化速度が観察されたことから、オゾンによる不活化はウイルスのカプシドの損傷から生じている可能性が高いことが示唆された。

第8章では、低圧紫外線ランプ(LPUV)および中圧紫外線ランプ(MPUV)によるウイルスの不活化について研究している。ポリオウイルスおよびアデノウイルスを対象とした。LPUVを用いた場合、1000mJ/cm2までの照射において、EMA処理の前後で核酸定量値が変わらなかったことから、カプシドの損傷は起きていないと考えられる。一方、MPUVを用いた場合には、特に300mJ/cm2以上においてはポリオウイルスおよびアデノウイルスともにEMA処理により核酸定量値が下がる結果が得られた。このことは、EMAが核酸に結合可能であったことを示しており、EMAが通過することが可能な損傷がウイルスのカプシドに生じていたことを示すことから、MPUVによりウイルスのカプシドが損傷したことが明らかとなった。次に、MPUVに対して様々な光学フィルタを合わせ、ことなる波長を持つ光によりウイルスの損傷を調べている。EMA処理による核酸の定量値低下は、230~240nm付近の波長の光が卓越している場合に大きいという結果が得られ、230-240nmの光がカプシドの損傷に効果があることが明らかとなった。

第9章は総括であり、本論文の結論および今後の展望について整理して示している。

本論文では、ウイルスの不活化に関して長らく不明であったオゾンによる消毒の不活化速度、および、塩素消毒、紫外線消毒ならびにオゾン処理におけるウイルスの核酸の損傷およびカプシドの損傷に関する研究を行い、重要な成果を上げている。このように、本論文は、水の微生物学的安全性を確保するための工学的な方法を体系的に評価するために重要な新たな知見をまとめたものであり、都市環境工学の学術分野に大いに貢献する成果である。

よって本論文は博士(工学)の学位請求論文として合格であると認められる。