- 學位論文
台灣地區Metapneumovirus 之培養及鑑定研究
Isolation and Identification of Human Metapneumovirus in Taiwan
摘要
在2001年於荷蘭的研究學者，首次經由具急性呼吸道感染症狀的嬰兒及小朋友的檢體中分離出human Metapneumonvirus (hMPV)， 之後在世界各地也相繼被發現。本次研究經由具臨床呼吸道感染症狀的檢體中，首次分離出屬於台灣本地的hMPV。 從2003年12月至2005年4月，總共收集4726呼吸道檢體，利用LLC MK2細胞作病毒培養來篩檢出hMPV病毒。其結果共分離出8 (0.17%) 株病毒，並藉由基因序列分析比對F Gene 及N Gene了解其分型狀況，其中A1 Subtype 2株、B1 Subtype 4株及A2及B2 Subtype各 1株。 根據觀察細胞病變(CPE)時，發現其hMPV在LLC MK2上所產生CPE形態有二種，包括了細胞變圓，局部細胞剝落以及細胞產生融合的現象。從接種至出現CPE的時間平均約20天(15-25天)。研究中發現培養基的pH值是CPE產生的主要因素，當hMPV在pH7.0及7.5的培養基培養時可產生相當明顯的CPE。為了找尋對hMPV更敏感的細胞株，本研究由LLC MK2細胞中選植(subclone)出7株細胞，發現當hMPV以pH7.0與7.5培養基下培養時，選殖細胞產生的CPE(3天)較未經選殖LLC MK2細胞(10天)更為明顯。 我們利用血清學的方法來調查hMPV在台灣的盛行率。一共收集116個血清檢體，年齡層由小於1週的新生兒至84歲的成人，利用免疫螢光染色法測量出血清中對hMPV的抗體。根據初步結果，在小於6個月的嬰兒抗體陽性達100% ，於6個月到1歲半為最低達60%，而青少年至成年(≦40歲)以上抗體陽性率逐漸上升達100%。於未來仍需收集更多檢體及深入的研究，以了解hMPV在台灣地區引起疾病感染情形及流行病學上的分佈。
關鍵字
間質性肺炎病毒並列摘要
Human metapneumovirus (HMPV) was first identified in 2001 in The Netherlands from infants and children with acute respiratory tract disease and is now recognized to be worldwide in prevalence. This is the first report that human Metapneumovirus (hMPV) were isolated from clinical specimens and associated with respiratory tract disease in Taiwan. The hMPV were screened by culture in LLC MK2 cell from 4726 respiratory specimens collected from Dec. 2003 to April 2005 in Taiwan. Eight (0.17%) hMPV strains were isolated and comparison of aligned F gene and N gene sequence were divided into 2 subtype A1, 4 subtype B1, and one each for A2 and B2 subtypes. Our own observations indicate that hMPV were 2 different morphologies of CPE in growth LLC MK2, consisting of rounded cells, focal plaques and with cell fusion. The CPE was apparent after a mean incubation time of 20 days (15–25days). The key element of observation of CPE is pH of the medium. When hMPV culture in pH 7.0 and 7.5 medium, we can obtain obvious CPE. In search of a cell line more susceptible for hMPV, we generated 7 subclones of LLC MK2 cell, CPE produced more prominent in LLC MK2 subclones (3 days), compared to LLC MK2 (10 days) when culture in pH7.0 and 7.5 medium. Serology tests were performed to investigate the prevalence of hMPV in Taiwan. The 116 serum samples from children aged from less one-week new bone babies to 84 years adults were tested for the presence of hMPV antibody using an indirect immunofluorescence assay. Preliminary result indicated that all baby (less than 0.5 years old) had hMPV antibody. After than, the seropositive rate decreased to 60% in children from 0.5 to 1.5 years old. The seropositive rate increased gradually to 100% from adolescent to adult (≦40 years old). Further prospective studies are required to define the epidemiology and the extent of hMPV infection in Taiwan.