腸病毒71型為不具外套膜之正股RNA病毒，在分類上為family Picornaviridae。腸病毒71型感染會出現特殊的臨床表徵，例如手足口症 (hand, foot, and mouth disease, HFMD) 或疱疹性咽唊炎 (herpangina)；有時則會出現嚴重神經性症狀。腸病毒71型在1969年第一次被分離鑑定於美國加州，之後，在世界各地皆有疫情傳出。其中，1998年在台灣爆發有史以來最嚴重一波腸病毒71型疫情，造成78嬰幼兒死亡，此波流行主要以基因型C2為主；之後腸病毒71型仍在台灣造成流行，在1998至2002年間，主要流行的基因型為B4；而至2004-2005年間，主要流行又轉變為基因型C4；而2008年在台灣則又有另一波以B5為主的流行。觀察過去近十年腸病毒71型在台灣之流行病史可發現，約每三到五年便有一波較大規模的流行，並且之間伴隨著基因型的轉變，主要是由基因型B及C交替出現。在台灣約有70%以上的人口體內具有腸病毒71型之中和抗體，許多研究也發現，抗體免疫反應是主要保護宿主對抗病毒的方式。病毒因受到宿主體內之中和抗體的壓力而產生突變，若這些突變位在病毒之殼蛋白 (capsid protein)，便會影響病毒之抗原性 (antigenicity)。我們也發現在每波不同基因型腸病毒71型之間，存在有抗原性之差異，進一步，我們想了解其抗原決定之位點。由於中和抗體效價較可反應病毒之抗原性，我們利用一免疫分析平台─結合微量中和試驗 (microneutralization test) 及酵素免疫吸附分析法 (enzyme-linked immunosorbent assay, ELISA) 測定健康者血清之中和抗體，發現對抗基因型B4之N7008-TW99病毒株得到較高之中和抗體效價；但對抗B5之N1745-TW08病毒株所得到的效價則較低。在比較基因型B4及B5病毒之VP1胺基酸序列後，發現其中有三個位點不同，分別位在VP1之第98、145及164胺基酸。在建構感染性克隆N7008-TW99 Inf及N1745-TW08 Inf後，則進行site-directed mutagenesis以得到帶有突變位點之infectious clones；進一步發現VP1第98胺基酸突變對於中和抗體效價有較大影響，VP1第145及164胺基酸雙點突變也可影響中和抗體效價。顯示VP1第98胺基酸是主要影響B4及B5病毒抗原性之位點。腸病毒71型抗原性之研究無論在致病機轉的探討或疫苗之研發上皆可提供一些重要訊息。

Enterovirus 71 (EV71) is a non-enveloped RNA virus, belonging to family Picornaviridae. EV71 causes many clinical presentations such as hand, foot, and mouth disease (HFMD), herpangina, and sometimes severe neurological complications. EV71 has resulted in several large outbreaks all over the world since the first report known as EV71 genotype A in California in 1969. In 1998, EV71 burst out the biggest outbreak in history in Taiwan and caused 78 fatalities. This pointed out that EV71 infection is an urgent issue. The major EV71 genotype in 1998 outbreak was C2. During 1998 to 2002, the major genotype was B4. However, the dominant genotype switched to C4 during 2004-2005. But in 2008, another outbreak was identified to be genotype B5. According to the findings, it showed that several different EV71 genotypes circulated from year to year in Taiwan. Most studies suggested humoral immunity played an important role in host against EV71 infection so that we proposed that neutralizing antibodies (NT Abs) existing in host may become a survival pressure for viruses. This pressure may select viruses and result in mutations. Therefore, we suggest that the antigenic variations may result from the change of different genotypes circulations in Taiwan. In this study, an ELISA-based microneutralization test assay was established to investigate the variations of serum neutralizing antibody titers which may suggest the change of viral antigenicity. We found that one serum with high NT titer against genotype B4 virus (N7008-TW99), however with low NT titer against genotype B5 (N1745-TW08). Sequence analysis of these two virus strains revealed that there were three different amino acids at position 98, 145 and 164 of structure protein VP1. Next, EV71 infectious clones of both viruses were constructed and site-directed mutant viruses were obtained. The result showed that amino acid position 98 in VP1 played the major role in viral antigenicity. In addition, the double substitutions at amino acid position 145 and 164 in VP1 could also affect NT titers against viruses. In conclusions, the amino acid substitutions of VP1 region resulted in the change of sera NT titers and played a role in viral antigenicity. These results might provide valuable information for pathogenesis and vaccine development of EV71.

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