- 學位論文
北台灣海水諾羅病毒之分子調查與水質評估
MOLECULAR INVESTIGATION OF NOROVIRUSES AND ASSESSMENT OF SEA WATER QUALITY IN NORTHERN TAIWAN
摘要
諾羅病毒﹙Norovirus，NV﹚是造成急性腸胃炎常見病原之一，而諾羅病毒主要利用水為媒介以糞口途徑傳播，因此必須有效地監測水環境諾羅病毒是否存在。本研究首先對於北台灣地區主要遊憩海域進行監測與分析，了解諾羅病毒在遊憩海域之分布情形，最後將針對近年台灣地區環境與臨床諾羅病毒之病毒株加以調查，並探討水環境與諾羅病毒傳播之關聯性。 本研究以逆轉錄-半巢式聚合連鎖反應﹙Reverse transcription - seminested PCR﹚，270 個糞便檢體中NV Genogroup I和Genogroup II陽性數分別為23個和38個。經由RT-PCR及半巢式-PCR雙重檢測分析確實提高諾羅病毒陽性檢出率，NVGI提高7.41％，NVGII提高7.40％，而配對 t-檢定分析後亦証實有顯著性的差異（p＜0.05）。 本研究為建立環境水體諾羅病毒濃縮方法，評估十種不同濾膜與濃縮流洗程序之組合，最佳的方法為添加金屬鹽類離子及調整pH值之前處理水樣，再以Nitrocellulose membrane 配合H2SO4-NaOH流洗法，並經由 MPN-RT-PCR﹙Most probable Number RT-PCR﹚確認諾羅病毒相對回收率達到100.00%。 實際環境調查之結果，顯示台灣海水環境已受到諾羅病毒污染，163個海水樣本有53個樣本呈現諾羅病毒陽性，NVGI和NVGII分別有22個和35個，其中有4個樣本是兩型別共同檢出。另外，再經過最大可能數MPN-RT-PCR半定量分析，諾羅病毒於環境之濃度分布為3-93 MPN/mL。而綜合國內外指標微生物大腸桿菌群、大腸桿菌和腸球菌之水質分級制度，經Kendall’s tau-b 相關分析，腸球菌水質分級制度與諾羅病毒（未檢出率）具高度相關，由此可知腸球菌分級標準除了應用於水質分級之參考也可用為評估諾羅病毒在暑期海水環境分布之指標。 在環境因子方面，經Pearson統計分析諾羅病毒與溫度呈現統計正相關﹙r=0.652，p＜0.05﹚，顯示當夏季人們於海灘的活動增加時，海水環境可能將受到相當程度的污染；pH值與諾羅病毒檢出呈現負相關性﹙r=-0.632，p＜0.05﹚，可能pH值較低有利於諾羅病毒存在而檢出頻率亦隨之上升；其他因子鹽度、潮差和雨量與病毒檢出率無顯著相關性。至於指標微生物濃度亦與病毒檢出之間無明顯相關性，可能所使用之指標微生物會受到太陽照射、溫度升高等因素，而無法反映海水受到諾羅病毒污染之真實狀況。 最後，臨床和環境之病毒分離株經由演化樹分析，NVGI與日本1999年、台灣2003年的流行株相近並歸類於GI-7型別；NVGII以GII-4為主要流行株與原型株（Lordsdale virus）相近，其中又可再分成兩群﹙1﹚重組株：可能來源自日本1998年的流行株﹙2﹚2006b：由環境和臨床所發現之病毒株與日本2006年、中國2007年之流行株相近。綜合以上所論，發現諾羅病毒確實存在於台灣北部臨床或海水環境中，並且可能在亞洲地區共同循環傳播。
並列摘要
Norovirus (NV) is considered as one of the most important agents causing acute viral gastroenteritis and is transmitted via fecal- oral route. Norovirus-related infections can occur to beach visitors by contrasting contaminated recreational waters. The aims of this study were (1) to validate the most efficient method on viral particle recovery; (2) to monitor Norovirus and some indicator microorganisms in the sea water for one year in the north-eastern Taiwan; and (3) to compare the isolated Norovirus strains, from the environments and those prevailing in clinics, using phylogenic analysis. Among ten filtration combinations, adsorption on nitrocellulose membrane, followed by acid rinse consistently gave the best recovery efficiency (100.00％). In the use of RT-seminested PCR, fifty-seven of the 270 stool specimens were shown as positive, with 23 (8.52%) for NVGI and 38 (14.07%) for NVGII respectively. Compared to the RT-PCR results, the detection sensitivity was increased by approximate 5% in the addition of seminested PCR (p<0.05, Pair t-test). As for the field samples, NVGII was the predominant strain (21.47%) whilst NVGI had 13.50 %. According to the standard pollution classification system using total coliform, E. coli and enterococci as factors, the correlation between the presence of enterococci and detectable norovirus was significant with Kendale’s tau-b analysis (p＜0.05). Therefore, enterococci can be not only a suitable indicator for water quality but also an alternative factor to evaluate the distribution of Norovirus in sea water, especially in summer. The presence of Norovirus was significantly correlated to the temperature and negatively correlated with pH value of water samples. However, no correlation was found between the presence of Norovirus and the concentration of microbial indicators. This may due to some indirect factors, such as the sunshine exposure and increasing temperature. According to the phylogenetic analysis, NVGI isolates were belonged to GI-7 group and the clinical and the environmental strains were more closely related to Japan 1999 and Taiwan 2003 outbreak. NVGII isolates from sea water samples were belonged to GII-4(2006b). All isolates either from stool specimen or sea water samples were separated as two groups (Recombinanted strain and 2006b). The recombinanted GII strains were grouped to GII-4 according to the RdRp gene and closely related to Japan 1998 prototype (Saitama U1). Furthermore, the other group, GII-4(2006b), was more closely related to the predominant strains in Japan 2006 and China 2007 outbreak. To sum up, the GII-4(2006b) strains were cocirculated in clinical patients and in sea water in Northern Taiwan. The opportunistic Norovirus may spread in Eastern Asia.