海水環境腺病毒調查與分析之研究

THE INVESTIGATION AND ANALYSIS OF ENVIRONMENTAL ADENOVIRUSES IN COASTAL WATER

周子崴(Tzu-Wei Chou)

指導教授 ： 陳建先

大同大學/工程學院/生物工程學系所/碩士(2007年)

摘要

腺病毒在台灣經常造成呼吸道感染、結膜炎與腸胃炎等感染流行，在世界各地皆有因水體受到腺病毒的污染而造成公共衛生危害的實例發生。因此本研究目的為建立一套適用於海水腺病毒回收之濃縮流洗技術，再以此最佳病毒濃縮方法搭配分子生物法與細胞培養法進行實際田野調查，以探討遊憩海域腺病毒分布與其感染風險性，並對於各項腺病毒檢測方法進行評估。
評估九種不同濾膜與濃縮流洗程序組合之海水腺病毒濃縮過濾技術，以調整pH與添加鎂離子之前處理水樣，先以硝酸纖維膜過濾濃縮，再以H2SO4/NaOH流洗法，經由MPN-PCR (Most probable Number PCR)和TCID50 (50% Tissue culture infectious dose)兩種定量法分析病毒相對回收率均達100%為最佳。而三種常用環境腺病毒定量方法(MPN-PCR、TCID50與Plaque assay [plaque forming infective unit] )其腺病毒定量之關係分別為1 MPN=1.73 TCID50 =1.55 PFU。然而使用ICC-PCR( Integrated cell culture-PCR )卻可於24小時檢測出0.1MPN/ml濃度之腺病毒。
本研究於實際田野調查結果顯示北台灣海水環境確實受到腺病毒污染，環境中腺病毒亦可能具有感染性。本研究亦證實海水環境中之腺病毒之檢出率不會受到水溫17.1℃-30.7℃變化的影響，但會受到部分地區潮位差異以及降雨的影響，並且指標微生物中大腸桿菌及大腸桿菌群可以反應腺病毒污染海水現況。由於環境與臨床檢體皆分離出ADV-3和ADV-41兩型腺病毒，經由核酸定序分析亦顯示環境分離株與臨床分離株核酸序列具有一致性，由此推論台灣地區腺病毒疫情與海水環境中之腺病毒有傳播之關聯性存在。因此海水環境進行腺病毒檢測時，分子檢測方法以A549細胞進行病毒培養再以半巢式-PCR(Seminest-PCR)進行ICC-PCR分析為佳。
而台灣腺病毒分離株特性為分蔥(Shallot)對ADV-41與ADV-3之50%感染抑制濃度(EC50)分別為733.9 與1137.6 mg/l，並且對此二型腺病毒自感染同時至兩小時內有最佳之抑制效果，另外檞皮黃酮(Quercetin)之濃度33.0及33.6 mg/l對ADV-41與ADV-3亦可達到50%感染抑制，其他蔥屬蔬菜如大蒜、洋蔥和青蔥於960mg/l濃度下，對ADV-41及ADV-3產生0.8-20.7%與1.8-16.4%之感染抑制效果。

關鍵字

環境調查 ； 腺病毒 ； 海水

並列摘要

Enteric adenoviruses generally spread through faecal-oral route but the importance of viral transmission by water is probably underestimated. Thus, a reliable and sensitive method for detecting low concentrations of adenoviruses is needed. The objectives of this study was to: a)develop a rapid and efficiency virus concentration method； b)evaluate PCR and cell culture for detection of adenovirus from water samples; c)compare the cell culture method with the seminest-PCR method in coastal water samples. Three different charges of adsorbing membrane filters and two elution methods were used in this study. Nine concentration method combinations, adsorption on nitrocellulose membranes followed by an acid rinse elution consistently gave the highest recovery efficiencies. Total 158 coastal water samples total positive detection rate were increased to 19.0% (30/158) when we applied seminest-PCR. The concentration of E.coli and total coliforms showed significant correlation with the presence of adenoviruses in coastal waters. It suggested that current recreational water quality standards could reflect the virus contamination in coastal water.
Sequencing analysis of the coastal water samples revealed that presence of adenovirus group F (type 41), group B (type 3), and group D, and most isolates belonged to serotypes 3 (83.3%). As to clinical samples, it appeared that there were sporadic cases for group B (type 3), C (type 2) and F (type 41). We compare the nucleotide sequence of adenoviruses both from clinical and environmental isolates by phylogenetic analysis. The result showed that environmental isolates were clustered as clinical isolates. The present study proved that the nucleotide sequencing analysis for studying epidemiological relationships between strains involved in human infections and in coastal water.
We had established a sensitive and accurate method for screening of anti-adenovirus agents using the MTT method. A549 cell line were used for adenovirus type 41 and 3 infection and examined for the anti-ADV activities of several kinds of Allium vegetables. The antiviral concentration of 50% effectiveness(EC50) of shallot at adenovirus type 41 was 733.9mg/l and the EC50 of shallot at adenovirus type 3 was 1137.6mg/l. According to the results of this study,the mode of action of shallot is derived from inhibition at an early stage of viral replication after infection.

並列關鍵字

INVESTIGATION OF ENVIRONMENTAL ； ADENOVIRUSES ； COASTAL WATER

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