G9輪狀病毒基因變異與血清抗體之分析

Abstract

輪狀病毒是全球造成嬰幼兒嚴重腹瀉、脫水甚至死亡的最重要致病原之一。輪狀病毒的變異性高，原因之一為病毒的RNA聚合脢缺乏矯正錯誤的功能而產生點突變；另一個重要因素與輪狀病毒之基因體呈片段狀有關，當兩種病毒同時感染一個細胞時，會產生基因重組 (reassortment) 之現象。依據病毒顆粒最外層的VP7和VP4兩種殼蛋白質，可將輪狀病毒分為22種G基因型和31種P基因型，其中G1P[8]、G2 P[4]、G3 P[8]與G4 P[8]是四種常見的輪狀病毒。從90年代中期開始，許多國家陸續分離出G9輪狀病毒，該病毒株已成為全球第五個主要流行的病毒株。臺大醫院在2000年首度發現四個G9輪狀病毒陽性檢體，之後便持續檢測出G9病毒株，到2002年，更成為該年主要流行的病毒，之後雖然G9病毒所佔比例有下降，但2000-2005年輪狀病毒陽性檢體中，G9病毒株所佔的數量仍是最多，G9病毒確實已在台灣地區流行。 對本地的G9病毒進行分析，大多數的病毒株屬於G9P[8]、VP6第二基因群、長型電泳型，只有五個病毒株屬於G9P[4]、VP6第一基因群、短型電泳型。針對VP7、VP4、VP6、NSP4、VP2及VP3基因進行序列比對及種系分析，發現本地G9輪狀病毒的VP7基因皆屬於分支III，且與最近國外流行的G9病毒在核酸與胺基酸序列相似性極高 (>98.6%)，在其他的基因片段，本地G9病毒株歸類在多個分支顯示變異性較大，與國內其他G血清型病毒株較相似。推測本地的G9病毒株很可能是經由國外進來的G9病毒與國內其他G血清型病毒株發生基因重組而產生。對本地代表性G9病毒株01TW591進行全基因體種系分析，其基因組合為G9-P[8]-I1-R1- C1-M1-A1-N1-T1-E1-H1 (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4- NSP5)，屬於Wa基因群的病毒，與2000年以後出現的病毒株關係較接近。 G9病毒已普遍存在於世界各地，到底有多少人曾經感染過G9病毒並不清楚，偵測血清型特異的抗體，將有助於了解個體是否感染過G9病毒。本研究利用中和試驗偵測G9輪狀病毒流行前、後血清中的抗體變化情形，比較1999年與2009年的孩童，發現G9病毒流行後，G9中和抗體有增加的情形，然而不論是1999年或2009年，個體針對G1、G3、G9病毒的中和抗體效價有相關性，很可能是因為有相同的VP4抗原造成交叉反應的現象。因此利用重組桿狀病毒只表現VP7蛋白質以去除干擾，並建立酶連試驗法來偵測血清型特異的抗體；但結果與中和試驗比較，兩者無顯著相關性。 本地流行的G9輪狀病毒株之基因變異性大且複雜，持續監控病毒的變化及是否有新興病毒的產生，並了解病毒株在族群中的感染情形及體內抗體呈現之狀況，將有助於未來控制輪狀病毒的流行。

Rotavirus infection is the most important cause of severe, dehydrating gastroenteritis among children worldwide. Rotaviruses have been characterized with high genetic variation. The viruses evolve through point mutation and genetic reassortment. According to the genetic and antigenic diversity of the two outer capsid proteins VP7 and VP4, rotaviruses are classified into G- and P-types. Globally, different surveys indicate that G1P[8], G2P[4], G3P[8] and G4P[8] are the most common G and P combinations encountered in human infections. Since the mid-1990s, novel G9 rotaviruses have been detected in many countries, suggesting that G9 is the fifth globally important serotype. In National Taiwan University Hospital, G9 rotavirus first appeared in 2000 with 4 cases, and was continuously detected until now. Most of the G9 strains belonged to G9 P[8], VP6 genogroup II, and long electropherotype, except five belonged to G9P[4], VP6 genogroup I, and short electropherotype. Phylogenetic analysis of the VP7, VP4, VP6, NSP4, VP2, and VP3 genes of representative local G9 strains showed that the VP7 genes shared a high degree of identity (>98.6%) to overseas G9 rotaviruses detected recently. The other genes were more variable and more closely related to those of local rotaviruses of other G types, suggested that local G9 rotaviruses possibly had evolved through reassortment between overseas G9 strains and local circulating rotaviruses of other G types. Based on a full-genome classification system, a representative G9 strain, 01TW591, was shown to possess the typical Wa-like genotype constellation: G9-P[8]-I1-R1-C1-M1-A1-N1-T1- E1-H1 (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5). G9 rotaviruses have been widely spread around the world, how much is the percentage of people infected with G9 virus is still unknown. To determine serotype-specific antibody, and then understand if the individuals infected with G9 rotavirus before, is importanat. Antibody titer from serum samples collected before and after G9 rotavirus epidemic was determined by neutralization assay. Comparing the antibody titer between the sera collected from the similar age populations in 1999 and 2009, G9 antibody titer was higher in the sera collected after G9 rotavirus epidemic. There were good correlations among G1, G3, and G9 antibody titers, which was possibly due to the cross-reactivity induced by VP4 epitopes. To reduce the interference of VP4 protein, an ELISA method using baculovirus expressed VP7 protein was used to determine serotype-specific antibody. The results were compared with those of neutralization test, but no significant correlation was observed. Local G9 rotaviruses had various genetic compositions and possibly had evolved through reassortment. Therefore, it is worthwhile to continuously monitor genetic relatedness among circulating strains and understand the antibody changes in rotavirus infection, which would be helpful to control the infections in the future.