校園廢水及回收水中病毒族群及大小分析

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题名:	校園廢水及回收水中病毒族群及大小分析
其它题名:	Surveillance of viruses diversity and abundance in MBR-treated reclaimed water
作者:	林至榮 Chih-Rong Lin
贡献者:	郭獻文 Hsion- Wen Kuo 環境科學與工程學系
关键词:	高通量定序;水中病毒濃度;腸道病毒;校園廢水;回收水;宏基因定序;即時定量聚合?鏈鎖反應 Adenovirus;hepatitis A virus;norovirus;enterovirus;high throughput screening;metagenomic;Real-time PCR
日期:	2016
上传时间:	2017-07-17T06:04:24Z (UTC)
摘要:	民生廢水中含有己感染者所釋放的致病病毒，雖然透過汙染控制技術可以大幅降低汙染物濃度，但是回收水依然可以發現致病病毒的存在，而目前台灣的回收水供應於景觀澆灌使用，因目前的汙染控制技術還未能掌握水中致病病毒的動態，使得回收水的使用範圍還屬於非飲用的階段而未能突破，然而使用回收水是現代補足缺水景況的一大趨勢，能夠指出廢水及回收水中的衛生問題有助於提升使用者的安全。有鑑於水質的細菌指標不足以代表實際微生物汙染物的族群，而根據文獻報告指出腸道病毒(例如人類腺病毒,HAdV)更適合代表微生物汙染的指標，使得本研究想探究腸道病毒在回收水中族群的大小。本研究目的是利用宏基因定序及即時定量PCR 調查校園廢水(CWW)及其經薄膜生物反應器(MBR)處理的回收水(RCW)中之病毒族群組成及大小，結果顯示宏基因定序產生的資料量為1.87×109-3.19×109 raw reads，接著透過de novo assembly 組裝成contigs 後比對NCBI GenBank 物種基因庫，可以比對到之Hits 有10.8-12.7％ contigs(可以比對到已知的物種Hits)以及未知的contigs 序列(No hits)約90%，CWW 和RCW 中病毒族群共有199 species 病毒家族，其中噬菌體(Phage)是最主要的群體為75.48-79.71%，包括Myoviridae、Siphoviridae、Podoviridae、Inoviridae、unclassified phage等病毒科；總病毒族群的人類病毒(human virus)為2.4-3.2 %，包括反轉錄病毒(RtV)、諾羅病毒(NoV)及C 型肝炎病毒(HCV)會造成人類的皮膚、呼吸系統及腸胃炎等疾病。在病毒族群定量方面，以即時定量PCR 偵測四個月份的腸道病毒的濃度，發現HAdV、HEV 及NoV-gii 均在CWW 和RCW 樣品中測定中分別出現濃度6.80×103-9.75×106、8.49×104-6.51×106 及1.84×104-1.37×107 virus particles/ L，一般而言傳染病有季節性的特徵例如腸道病毒在夏季為流行感染的季節，而在本研究結果中並未發現腸道病毒有季節性的特徵，而本研究結果證實CWW 及RCW 中病毒的族群及大小之複雜且不具週期性，而為了水質的衛生安全需要監測多種的腸道病毒的項目，制定多種腸道病毒共通的分析方法會使水中致病病毒的管理更佳安全。 Pathogenic viruses are a group of microbial contaminants frequently occur in municipal wastewaters or even in reclaimed waters. Although practice of pollution control techniques could reduce the concentration of these microbial pollutants, hygiene and safety problems about discharge or reuse treated wastewater are still being questioned. Reclaimed water is most commonly used for nonpoTable (not for drinking) purposes, such as agriculture, landscape, public parks, and golf course irrigation. Given that bacterial indicators are insufficient to represent actual microbial contamination conditions, some human enteric viruses (e.g., human adenovirus, HAdV) have been considered as more suiTable pathogenic indicators. The objectives of this study were to investigate viral diversity based on metagenomic sequencing and to assess viral concentration using quantitative real-time PCR assays for a campus wastewater (CWW) and its MBR-treated reclaimed water (RCW) samples. Results showed that more than 1.87×109-3.19×109 raw reads were obtained for each sample library which were used for further contig assembly by using the CLC Genomics Workbench. Majority (96.4%)of the assembled contigs had a size larger than 300 nt. About 10.76-12.65% of the assembled contigs were hit with sequences within NCBI GenBank and more than 90% of hit contigs were identical with some viral genomic sequences for both CWW and RCW samples. Among the identified viral community, bacterial phages were the most dominant group (76.51% and 77.60% for CWW and RCW samples, respectively) including phages of the Myoviridae, Podoviridae, and Siphoviridae families for both CWW and RCW samples. Besides, 2.44 - 3.23% of revealed viruses were related to human infections for CWW and RCW samples, accordingly; including Retrovirus,Norovirus, and Hepatitis C viruses responsible for skin diseases, respiratory diseases,and gastroenteritis. Additionally, human adenovirus (HAdV), human enterovirus (HEV),and norovirus group Gii (NoV-Gii) were detected in all CWW and RCW samples by real-time PCR assays with concentrations ranged from 6.80×103 to 9.75×106 viral particles/L, from 8.49×104 and 6.51×106 viral particles/L, and from 1.84×104 and 1.37×107 viral particles/L, respectively; while norovirus group Gi (NoV-Gi) were not detected in all samples. Overall results demonstrated diverse viral communities wereoccurred in both campus wastewater and its MRR-treated reclaimed water providing further viral pollution information of municipal wastewater for public health awareness.
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