Human enteric viruses have been recognized as one of the major causes of waterborne outbreaks which have been reported worldwide in both developed and developing countries. Enteric viruses related to waterborne diseases include noroviruses, enteroviruses, hepatitis viruses and adenoviruses. Viruses are excreted in feces of infected individuals in high concentration and gathered in sewage. Viruses in sewage can contaminate recreational water body and hellfish harvesting area in coastal water to pose a great risk to the public health. Molecular methods have recently been used as a tool to detect viruses in the environment but most studies provided only qualitative data i.e., presence or absence of the viruses, which lead to the limited information on their behaviors.

The main point sources of viral contamination in urban coastal water are combined sewer overflow and wastewater treatment plant, but actual effect of combined sewer overflow and effluent as well as the degree to which they contribute to viral contamination in coastal water has not been well characterized. Thus, this research aimed to evaluate the occurrence and behavior of human enteric viruses in urban coastal water which contaminated by combined sewer overflow and effluent. The target viruses in this study include norovirus G1, norovirus G2, enterovirus and adenovirus. The occurrence and behavior of microbial indicators was studied in parallel with these viruses. The microbial indicators examined include (i) conventional fecal pollution indicators (total coliform, fecal coliform, E.coli), (ii) enterococcus which is recently recommended fecal indicator in marine water, (iii) Clostridium perfringens spores, and (iv) two types of bacteriophage viz., FRNA bacteriophage and somatic coliphage, which recently has been proposed as viral indicators. The real-time PCR was used to quantify concentration of viruses. Plate culture assay was used to determine concentration of microbial indicators.

In Chapter 4, the profile of viruses in sewage and their behaviors during passing through wastewater treatment processes was investigated at two wastewater treatment plants (Morigasaki and Ariake wastewater treatment plants) in Tokyo. Combined sewer overflow can be represented by raw sewage in wastewater treatment plant. The raw sewage, effluent before chlorination and final effluent after chlorination were collected on monthly basis for one year. The results showed that human enteric viruses were detected in all sewage samples. The most prevalent virus detected was adenovirus. Seasonal pattern was observed in case of norovirus G1 and norovirus G2 in which their concentrations were high in winter and low in summer. The concentration of enterovirus and adenovirus in sewage was relatively constant throughout the year. An average of overall removal efficiency in both wastewater treatment plants for all types of viruses tested, C. perfringens spores, bacteriophages, and bacterial indicators were 2.4 - 3.6 logs, 2.3 logs, 3.4 - 3.6 logs, and 4.5 - 5.5 logs, respectively. Obviously, wastewater treatment plants were more effective in removing bacterial indicators than enteric viruses. The results indicated that the behavior of bacterial indicators does not adequately explain virus removal in wastewater treatment plants. Among viruses, norovirus G1 was the most resistant virus whereas norovirus G2 was the least resistant virus against biological treatment and chlorination. The difference of ratio of concentration of resistant microbes (viruses, bacteriophage or spore) to fecal coliform between sewage and wastewater treatment plant effluent gave useful information for tracking the viral pollution sources in coastal area.

Chapter 5 was focused on distribution and persistence characteristics of viruses following the combined sewer overflow discharge to coastal water under laboratory controlled condition. The vertical distribution of solid-associated viruses through sedimentation process (60 minute settling times) in column experiment was studied. The results showed that the amount of viruses existing in solid-associated form more than free-suspended form, except adenovirus which tended to exist more in free-suspended form. Viruses in solid-associated form were increased in saline water. The percentage of all type of viruses in solid-associated form was in the range of 50 - 76% at salinity 0% condition while it was in the range of 62 - 96% when salinity increased to 1% or 2.5%. The changing in concentration of viruses was governed by their persistence to environmental conditions. Norovirus G1 was the most persistent virus whereas norovirus G2 was the least persistent virus among all type of viruses tested in dark condition. Light irradiation of metal halide lump accelerated degradation of viruses. The influence of salinity on disappearance of viruses was observed greater in the presence of light than dark condition. Solid particles reduced degradation of viruses under light exposure and also in dark condition.

Chapter 6, field monitoring was carried out to study the occurrence and behavior of viruses in coastal area of Tokyo Bay as well as to identify source of viral contamination. Water and sediment samples were collected in three sampling sites in Tokyo Bay on monthly basis for one year. The most abundant virus found in Tokyo Bay was adenovirus. The prevalence of noroviruses was observed in winter. The results showed that viruses were present even when fecal coliform level was below the Japanese recreational standard. The elevated level of viruses and microbial indicators in Tokyo Bay occurred after heavy rainfall event. The level of virus concentration was not only depended on rainfall quantity and duration of dry days but also persistence of viruses. The persistence of microorganisms in Tokyo Bay had the same tendency in wastewater treatment plant. The order from greatest persistence to the least persistence was C. perfringens spore > viruses and bacteriophages > bacterial indicators. The mean concentration of viruses and microbial indicators in sampling sites in Tokyo Bay was higher than mean concentration in effluent from the wastewater treatment plants. The ratio between concentration of resistant microbes (viruses, bacteriophage or spore) to concentration of fecal coliform in Tokyo Bay samples was increased because fecal coliform was degraded more rapidly than resistant microbes. The results of field monitoring demonstrated that viral contamination in coastal zone in Tokyo Bay was governed by combined sewer overflow rather than daily effluent discharge from wastewater treatment plant. The impact of direct combined sewer overflow discharge was observed in both water and sediment in one of the three sampling sites, which is near the outlet of combined sewer overflow.

In Chapter 7, the occurrence of enteric viruses in environmental samples in Indonesia and Vietnam, where epidemiological data on viral prevalence is unavailable, were studied and compared to the occurrence in Japan. The results demonstrated that novovirus G1 , norovirus G2, enterovirus and adenovirus were prevalent in Indonesia, Vietnam and Japan but hepatitis A virus was prevalent only in Indonesia. This result revealed that occurrence of enteric viruses varied from country to country. The concentration of viruses in surface water in Indonesia and Vietnam was greatly varied depended on level of contamination. Some surface water samples or floodwater samples contained enteric viruses with concentration similar to raw sewage in Japan.This would pose high risk of viral infection to people through contamination of drinking water or direct contact with waters. The result of Chapter 7 suggested that determining of occurrence of viruses in environmental samples is useful for assessing the prevalence of viruses in the community.

　本論文は、Occurrence and Behavior of Human Enteric Viruses in Urban Coastal Water(都市沿岸水中におけるヒト腸管系ウイルスの存在およびその消長)と題し、水系感染性のウイルスについて研究したものである。8章で構成されている。

　第1章では、水系感染症におけるヒト腸管系ウイルスについて概観し、ウイルスの自然水系への排出経路の一つとしての合流式下水道雨天時越流水の重要性を説明し、研究の目的および研究の構成を示している。

　第2章では、さまざまなヒト腸管系ウイルスの特徴、ならびにウイルスの感染経路について概観し、環境水中のウイルス測定に関する既存の知見をまとめ、さらに、さまざまな微生物の水中における残存性に関わるさまざまな要因について、既存の研究をまとめている。

　第3章では、本研究における調査対象について説明している。本研究で対象とした微生物は、ノロウイルスG1、同G2、アデノウイルス、エンテロウイルス、細菌ファージ2種類、糞便指標細菌3種類および嫌気性芽胞菌であり、水試料および底泥試料からのそれぞれの定量法を説明している。

　第4章では、一年にわたり東京都内の2箇所の下水処理場から流入下水と二次処理水、および塩素処理後の放流水を採取し、ヒト腸管系ウイルスおよび指標細菌を測定している。ヒト腸管系ウイルスの中ではアデノウイルスの濃度が高く、ノロウイルスは冬に濃度が高いこと、微生物の除去効率は指標細菌のほうがウイルスより高いという結果を得ている。また、ノロウイルスG1とG2を比較し、G2の方がG1よりも除去されやすいことを見出している。

　第5章では、海域における微生物の挙動を推定するため、流入下水と塩分濃度の異なる水とを混和して沈降試験を行っている。さまざまな微生物の水中微細粒子への吸着特性と沈降速度を比較し、ウイルスは浮遊性粒子に吸着しやすいこと、特にノロウイルスとエンテロウイルスは塩分濃度1％以上で浮遊性粒子に吸着しやすくなることを明らかにしている。また、沈降1時間後の上層の画分に対して10日間の残存試験を行い、ウイルスは嫌気性芽胞よりも残存しにくいが、糞便性大腸菌群よりは長く残存することを示した。ただし、ノロウイルスG2は指標細菌と同等の残存率であり、ウイルスの中でも残存性が異なることを見出している。

　第6章では、1年間にわたる毎月の東京湾における現地調査の結果を示し、考察している。合流式下水道雨天時越流水の影響を大きく受ける地点、下水処理水の放流口に近い地点、および人との接触が考えられる地点の3箇所を対象とした。ノロウイルスは冬季に高い濃度を示していること、先行晴天日数が短い方が微生物濃度が高い傾向にあることを見出している。また、腸管系ウイルスの糞便性大腸菌群に対する比率を流入下水と東京湾の試料とで比較し、腸管系ウイルスが糞便性大腸菌群より残存しやすいことを明らかにしている。

　第7章では、インドネシアのジャカルタ市ならびにベトナムのホーチミン市における水中のヒト腸管系ウイルスの測定結果をとりまとめている。都市内の水路や道路の冠水に大腸菌とともに多くのヒト腸管系ウイルスが含まれること、ジャカルタではA型肝炎ウイルスが高濃度で存在していることを明らかにしている。

　第8章は総括であり、本論文の成果を取りまとめて示してある。

　以上のように、本論文は、下水ならびに東京沿岸の水に含まれるヒト腸管系ウイルスを実測することによりその挙動を明らかにしたものであり、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と認められる。