南台灣二十年來腺病毒第三、四、七型分子流行病學研究

Molecular Epidemiology of Adenoviruses Type 3, 4, 7 over a 20-year Period in Southern Taiwan

李鴻梅(Hung-Mei Lee)

指導教授 ： 林貴香

高雄醫學大學/醫學院/醫學研究所/碩士(2006年)

https://doi.org/10.6832/KMU.2006.00063

摘要

腺病毒是造成嬰兒與兒童呼吸道感染的主要病因之一，至目前為止已有51種血清型被發現。依據腺病毒之生化特性、致癌性與DNA同質性等可分為A~F六種亞屬。腺病毒中的亞屬B、C、E常與急性呼吸道感染有關，尤其是嬰兒與兒童之急性呼吸道感染。在台灣的腺病毒研究中發現Ad3、Ad4與Ad7之流行與幼童嚴重的下呼吸道感染相關，故持續的進行腺病毒的監測是必要的。
本研究主要目的為瞭解(1)造成臺灣地區呼吸道腺病毒流行之血清型的概況，及(2)分析較容易造成嚴重呼吸道疾病的Ad3、Ad4、Ad7的基因型 (3)定序基因序列進行血清型分型及演化分析。(4)將病毒基因分型的結果與病患臨床資料作一分析統計。
1981至2006年分離到的腺病毒陽性株2500株，隨機選取1443株進行腺病毒的研究。血清分型是利用PCR及PCR-RFLP方法；基因分型使用RFLP方法分析88株Ad3，48株Ad7與24株Ad4。為了要瞭解Ad3、Ad4、Ad7之基因演化，定序腺病毒hexon 與fiber基因序列，利用DNAstar及MEGA 3.1軟體分析。另外調閱病歷，分析病患之年齡、性別、發病月份、臨床病徵與臨床診斷等資料。
腺病毒亞屬B(60%)為此研究期間最主要的血清型別，其次為亞屬C(29%)，再其次為亞屬E(11%)。在所有的血清型別中，Ad3在1981-2006年為兒童呼吸道疾病最常見之型別(除了在2001年時)。Ad4與 Ad7分別為1999與2001年主要流行之血清型。在Ad3的基因型研究中發現Ad3a、Ad3a1與Ad3a2三種基因型，在1999年以前的Ad3以3a為主，2000年以後以3a2所佔比例較多；在Ad4的基因型研究中只發現Ad4a基因型；在Ad7的基因型研究中發現Ad7a、Ad7b二種基因型，Ad7b為主要的基因型別。本研究定序腺病毒 hexon 與fiber基因序列進行分析：將部份hexon序列進行演化分析，Ad1-Ad7血清型於基因樹狀圖上可以明顯的分群；Ad3全長hexon基因序列分析可將Ad3a與Ad3a2分群；在Ad7 fiber基因序列分析則可將Ad7a與Ad7b分群。調閱病歷資料分析發現4-6歲(81%)為主要發病年齡群；男女比例為1.4：1；腺病毒流行季節是全年均可見；臨床診斷依序為急性扁桃腺炎(67%)，肺炎(21%)，急性氣管炎(20%)，急性胃腸炎(17%)，有些病患有2種臨床診斷。PCR-RFLP為鑑定腺病毒血清型一方便、正確與快速的方法；而RFLP為進行基因型分析的工具；結合PCR與序列分析為更加敏感的方法。因為若是分析的病毒DNA正好於RFLP的酵素切位突變，RFLP比對會有困難，而結合PCR與序列分析可以克服此問題。

關鍵字

腺病毒 ； 分子流行病學 ； 腺病毒基因型

並列摘要

Adenovirus is one of the important causes of respiratory infection in infants and children. Fifty one serotypes of human adenoviruses (Ads) have been classified in to six subgroups (A-F) based on their biological characteristics, tumorigenicity, and DNA homology. Ads in subgroups B, C and E are responsible for acute respiratory disease, especially in infants and young children. Since the Ad3, Ad4 and Ad7 epidemic in Taiwan have been associated with severe childhood lower respiratory disease, continuous surveillance was necessary for Ads related disease.
To characterize epidemiology features of Ads from 1981-2006, Ads serotypes distribution, genetic diversity of Ad3, Ad4 and Ad7, phylogenetic relationships of Ads were analyzed to understand the evolutionary process. To reveal the serotypes, genotypes changing and molecular epidemiology of Ad3, Ad4 and Ad7 from 1981-2006, their association with clinical manifestations was also studied.
From 1981 to 2006, a total 1443 virus strains were random selected from 2500 adenovirus isolated. Serotype analysis was performed by PCR and PCR-RFLP. Genotypes were determined by RFLP to analyze 88 selected strains of Ad3, 48 selected strains of Ad7, and 24 selected strains of Ad4. In order to understand the evolutionary relationships between Ad3, Ad4 and Ad7 strains, DNAstar and MEGA version 3.1 were used to construct phylogenetic trees and analysis differences. Medical records were reviewed retrospectively. Age, sex, seasonal distribution, cilinal manifestations and clinical diagnosis of patients infected with Ad3, Ad4 and Ad7 were analyzed. The major subgroup found was subgroup B (60%), followed by subgroup C (29%) and subgroup E (11%). Ad3 was the major type of respiratory infection during 1981-2006, except 1999 and 2001, while Ad4 and Ad7 were the predominant in 1999 and 2001, respectively. In genotype analysis of Ad3, three genotypes, Ad3a, Ad3a1 and Ad3a2 were found. Only Ad4a was found in the study period. In Ad7, two genotypes, Ad7a and Ad7b have been identified. Phylogenetic trees of Ad3, Ad4 and Ad7 were inferred based on hexon and fiber sequences. The phylogenetic analysis of Ad1-Ad7 nucleotides sequences based on partial hexon gene suggested that Ad1-Ad7 can be identified. Phylogenetic tree was inferred based on full length of Ad3 hexon sequences. Phylogenetic trees revealed that Ad3a2 can be distinguished from Ad3a and other foreign strains. Phylogenetic trees based on the Ad7 fiber demonstrated that Ad7a and Ad7b can be distinguished. In medical records analysis, children between 4-6 years old were the most common group of patients (81%) with respiratory adenoviral infections. The ratio of male to female was 1.4:1. In seasonal patterns of respiratory adenoviral infections, Ads was detected throughout the year. The most common presentation was followed by acute tonsillitis (67%), pneumonia (21%), acute bronchitis (20%) and acute gastroenteritis (17%). The result of this study demonstrated that PCR-RFLP analysis is a convenient, accurate, and rapid method for serotyping of adenoviruses. Genotype changing was revealed by RFLP. The combination of PCR and sequencing analysis is more sensitive, RFLP may fail in genotyping a virus in the presence of a point mutation on the restriction site, whereas sequencing analysis overcomes this disadvantage.

並列關鍵字

adenovirus ； molecular epidemiology ； genotype

參考文獻

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