北台灣地區G3型輪狀病毒之分子流行病學研究

Abstract

輪狀病毒(rotavirus)是造成全世界嬰幼兒腹瀉最重要的病因，目前尚無有效的治療方法，所以疾病的預防就顯得更加重要。而分子流行病學可以監控歷年來輪狀病毒基因的變異及演化，並可利用這些分析預估現有的疫苗是否有效，並為往後新型疫苗的研發提供依據。 輪狀病毒的結構可以分為三層，最外層由抗原蛋白質VP7和VP4所組成，中層由VP6組成，最內層的核心(core)含有11段雙股核糖核酸(dsRNA)，以及其他結構性蛋白質，此外，病毒尚有非結構性蛋白質，如NSP4等。早期利用ELISA對VP6、VP7、VP4進行分型，現在則多利用RT-PCR等分生的方法。依VP6抗原性的不同可將輪狀病毒分為A~E等群，人類大部分所屬的A群又可以分為亞群I和II；再依VP7、VP4基因型的不同，可區分為15種G型和27種P型；依NSP4的不同，可以分為A~E基因型；此外，將病毒RNA進行凝膠電泳，依第10和11段RNA移動的速度，可以分為RNA電泳型長型和短型。目前世界上最常見的輪狀病毒基因型為G1P[8]、G3P[8]、G4P[8]以及前幾年興起的G9P[8]，這些基因型通常也屬於亞群II、長型電泳型；而G2P[4]則大都屬於亞群I、短型電泳型。 本研究係針對台大醫院所收集來自北台灣1983年到2005年來的G3型輪狀病毒檢體 (絕大部分為P[8])，依其RNA電泳結果分類並挑選檢體，進行VP7、VP4、VP6、NSP4基因之序列及種系分析，結果列述如下。 (1) 在VP7基因的分析，可以區分為兩群，1983~1991年之早期檢體為G3-I型， 1997~2005年之晚期檢體則為G3-II型。 (2) VP4基因的分析則可以看到大部分1983~1990年之早期檢體屬於P[8]-I型，1990到1991年出現P[8]-II型，到了1997~2005年，P[8]-II型又再度持續流行。 (3) NSP4基因的分析可以分為三群，1985~1991年之早期檢體為B-I型，B-II型則包含1997和2001年的檢體，1999~2005年之晚期檢體則為B-III型。 (4) VP6基因方面， 1985~1991年早期檢體都是屬於subgroup II-1，然而晚期檢體則出現了兩種支型，一部分2002~2005年的檢體與早期檢體同屬subgroup II-1，然而剩餘的1997~2005年晚期檢體皆屬於subgroup II-2。 上述同一群彼此之間的基因及胺基酸相似度大多高達95~99%；胺基酸變異的分析方面，經比對1977年的日本參考株YO、北台灣早期和晚期檢體中輪狀病毒之VP7和VP4的抗原變異區以及NSP4的功能區域有發生變異，有可能晚期與早期輪狀病毒的抗原性發生了變異。 北台灣早期病毒株如何演化成晚期病毒株是一個有興趣的問題，推論可能發生基因重組(reassortment)、基因突變或是境外移入，本篇研究結果可為疫苗的研發提供重要的參考依據。

Rotavirus is the most important pathogen causing infantile diarrhea worldwide. Prophylaxis of rotavirus infection is important due to unavailability of efficient treatment. Monitoring genetic variation and evolution of rotavirus is essential for estimating the efficiency of developing vaccines and providing information for vaccine research. Rotaviruses are formed by three layers of proteins. The outer layer is composed of outer capsid proteins VP7 and VP4, and the middle layer is formed by VP6 protein. The inner core contains 11 segments of double stranded RNA genome. Rotaviruses are divided into groups A~G depending on the antigenicity of VP6. Most human rotaviruses belong to group A and are classified into subgroups I and II. Based on VP7 and VP4 genes, group A rotaviruses are further differentiated into 15 G types and 27 P types; according to NSP4 gene, group A rotavirus can be classified into genotypes A~E. Besides, according to the position of the tenth and eleventh gene segments on RNA polyacrylamide gel electrophoresis, rotavirus RNA can be differentiated into long or short eletropherotype. Globally, epidemiological studies of rotavirus revealed that the most common types include G1P[8], G3P[8], G4P[8], and emerging G9P[8], characterized by subgroup II and long electropherotype, and G2P[4] characterized by subgroup I and short electropherotype. This study investigated the genetic characteristics of G3 rotavirus genome in northern Taiwan from 1983 to 2005. G3 rotaviruses in northern Taiwan appeared in two major periods, early period (1983~1991) and late period (1997~2005). The VP7, VP4, NSP4, and VP6 genes of these Taiwanese G3 rotaviruses from the two periods were amplified by RT-PCR, and then sequenced, and phylogenetically analyzed. In the phylogenetic analysis of the VP7 genes, G3 rotaviruses can be classified into G3-I (1983~1991) and G3-II (1997~2005). For the VP4 genes, G3 rotaviruses can be differentiated into P[8]-I (1983~1990) and P[8]-II (1990~1991 and 1997~2005). For the NSP4 genes of Taiwanese strains, three lineages, B-I (1985~1991), B-II (1997 and 2001), and B-III (1999 ~2005) were found. For the VP6 genes, II-1 (1985~1991, 2002 ~2005) and II-2 (1997~2005) were identified. Both nucleotide and amino acid identities within the same lineages in each phylogenetic analysis were within the range from 95 to 99%. G3 rotaviruses in northern Taiwan were possibly revolved through point mutation, genetic reassortment, and some of the strains were possibly imported from other countries. The information provided by this study will be beneficial for future vaccine development.