南台灣近二十年來腸病毒之分子流行病學暨 單株抗體的製備與特性分析

A Two Decade Survey of Enterovirus as well as the Preparation and Characterization of Their Monoclonal Antibodies in Southern Taiwan

王珠鳳(Chu-Feng Wang)

指導教授 ： 林貴香

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

腸病毒屬於小RNA病毒科（Picornaviridae），腸病毒屬(Enterovirus)。腸病毒屬共有67個血清型，以病毒的基因型可將腸病毒屬分為5個種系(species)，分別為(1) Poliovirus(P1-3)、(2) Human enterovirus A(HEV-A,包含CA2~CA8, CA10, CA12, CA14, CA16, EV71)、(3) Human enterovirus B(HEV-B,包含P1~3, CA9, CB1~CB6, E1~E7, E9, E11~E21, E24~E27, E29~E33, EV69, 與評估中的EV73)、(4) Human enterovirus C (HEV-C,包含CA1, CA11, CA13, CA15, CA17~CA22, CA24)、(5) Human enterovirus D(HEV-D,包含EV68, EV70)。腸病毒的感染遍及全世界，主要是經由飛沫、糞口及接觸等途徑感染，感染所造成的疾病包括有：呼吸道症狀、手足口病、無菌性腦膜炎、腦膜腦炎、急性肢體麻痺、出血性結膜炎等。台灣於1998年爆發腸病毒大流行，當時造成幼兒高致死率，腸病毒七十一型是主要的病毒株造成400多位幼兒住院，78位因中樞神經感染而引起心肺衰竭死亡。其後幾年仍持續發生不同型的腸病毒流行。因此腸病毒早期診斷成為當前重要課題。但是近年來無法分型之非polio腸病毒(NPEV)正逐年增加，因此快速準確的鑑定與分型對腸病毒流行之掌握是刻不容緩的事。此外，多種有效的腸病毒單株抗體之開發對早期診斷是很重要的。因此本研究共分兩部分，第一部分在闡明過去20年來腸病毒的流行情況。第二部份在製備EV71的單株抗體，期能供EV71早期診斷。
將1980 ~ 2005年的腸病毒分離，藉間接螢光抗體試驗，中和試驗和RT-PCR及序列分析做鑑定分型。病毒分離以Vero, MK2, RD細胞株做病毒培養。並以市售單株抗體做 IFA來鑑定分型。無法分型之非polio腸病毒 (NPEV) ，萃取其RNA，放大其VP4基因加以定序分析比對，鑑定其型別。分析結果顯示在過去近20年來有多次腸病毒流行發生。E30為1993年的主要流行株 (295/387,76.2%) 。EV71 在1986年出現，並在1986, 1998, 2000, 2001年成為主要流行株。CA16是1998年的次流行株，並在2000, 2003成為主要流行株。科沙奇病毒B型則是CB1於1994, CB3於1999, CB4於2004, CB5於2002各為主要流行株。此外，EV70在1980, 1981和1983年有出現。CA24v在1985-1986, 1988-1989年有出現。EV70及CA24v都會引起急性出血性結膜炎。本研究第一部分的結果揭示台灣二十年來腸病毒之流行趨勢，將有助於腸病毒流行的瞭解及疾病監控。
EV71單株抗體藉融合瘤細胞技術製備，病毒大量繁殖及純化。經由皮下、腹腔內或直接脾臟注射免疫BALB/c小鼠。免疫完成後取脾臟細胞與骨髓瘤細胞 (NS-1) 融合成融合瘤細胞。共篩得4株融合瘤E211F, E211F2B, E410G2B, E611G2G。以ELISA、NT、FA、 WB等方法進行抗體篩檢及其特性分析。將融合瘤細胞注入BALB/c小鼠腹腔產生腹水,大量製造單株抗體。所得之單株抗體做其亞型分析(isotyping) 其中3株為IgG1 (E211F, E211F2B, E410G2B) ，E611G2G為IgG3。有關此4株單株抗體的專一性和敏感性之特性分析，尚待進一步與多種血清型之腸病毒做間接免疫螢光染色,以驗證其專一性，並與各年代不同基因亞群的EV71及以市售EV71單株抗體無法分型但序列分析結果為EV71之病毒株做間接免疫螢光染色,以驗證其敏感性。本研究第二部分的結果提供新的製備好之單株抗體，有助於EV71之快速正確之診斷。

關鍵字

腸病毒 ； 單株抗體

並列摘要

Enteroviruses are small RNA viruses of the Picornaviridae family, enterovirus genus. Enterovirus consists of 67 serotypes, consists 5 species by genotyping including (1) Poliovirus(P1-3)、(2) Human enterovirus A(HEV-A,CA2~CA8, CA10, CA12, CA14, CA16, EV71)、(3) Human enterovirus B(HEV-B,P1~3, CA9, CB1~CB6, E1~E7, E9, E11~E21, E24~E27, E29~E33, EV69,EV73)、(4) Human enterovirus C (HEV-C,CA1, CA11, CA13, CA15, CA17~CA22, CA24)、(5) Human enterovirus D(HEV-D,EV68, EV70) by genotype. Enterovirus infections are among the most common infections in the world. The diseases range from respiratory symptom, hand- foot- and- mouth disease (HFMD), aseptic meningitis, meningoencephalitis, acute flaccid paralysis(AFP) to acute hemorrhagic conjunctivitis(AHC). In 1998 a devastating outbreak of enterovirus occurred in Taiwan. EV71 was incriminated as the causative agent. More than 400 children were hospitalized, with 78 deaths due to central nervous system involvement and cardiopulmonary collapse. Outbreaks caused by different enteroviruses occurred in following years. Since then, early diagnosis of enterovirus infection became an urgent issue in Taiwan. However, the number of untypable non polio enterovirus (NPEV) increased during recent years. It is important to understand the trend of epidemiology of enteroviruses for the control measures of our government. Besides, a powerful panel of enterovirus mAbs is critical for the early diagnosis. To address this, firstly, we unravel the epidemiology of enterovirus in the past two decades. Secondly, we prepare mAb of EV71 for the early diagnosis.
Enterovirus isolates obtained from 1980 to 2005 were studied using IFA, NT and RT-PCR, and sequence analysis. Virus isolates were cultured in Vero, MK2 and RD cells and identified by IFA using commercialized mAb. VP4 region of the isolates were amplified and sequenced. The types of enterovirus were identified by sequence analysis. The results showed that several outbreaks occurred in the past two decades. Different enterovirus type was predominant in different outbreaks. E30 was predominant in 1993 outbreak. E11 was predominant in 2003 outbreaks(295/387, 76.2%). EV71 remerged in 1986, and became predominant in 1998, 2000, 2001 outbreaks. CA16 was the second major type found in 1998 and 2000 outbreaks , and was predominant in 2003 outbreaks. For the coxsackies B virus (CB), CB1 was predominant in 1994, CB3 in 1999, CB5 in 2002, CB4 in 2004, CB3 in 2005. Besides, EV70 was found in 1980, 1981 and 1983 outbreaks. CA24v was found in 1985-1986, 1988-1989 outbreaks. Both EV70 and CA24v cause acute hemorrhagic conjunctivitis. The results from the first part of this study revealed the epidemiology of EV in Taiwan. This is benefit to the understanding and control measures of EV outbreaks in Taiwan.
monoclonal antibodies (mAbs) of EV71 were prepared by hybridoma technique. Viruses were cultured and purified. BALB/c mice were immunized via intrasubcutaneous, intraperitoneal or direct injection into spleen. Immunized spleen cells were hybridized with NS-1 myeloma cells. Four clones of hybridoma cell line, E211F, E211F2B, E410G2B, E611G2G, were obtained by screening using ELISA, NT, FA and techniques. The monoclonal antibodies were mass produced by intraperitoneal injection of the clones into BALB/c mice. The isotypes of the three clones, E211F, E211F2B, E410G2B, are IgG1 isotype, and the other one, E611G2G, is IGg3. No cross reaction of the mAb (E211F) were found between EV71 and 13 other enteroviruses by FA. The results from the second part of this study offered new mAbs which were critical for the early detection of EV71.

並列關鍵字

monoclonal antibody ； Enterovirus

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