豬鐵士古病毒高度保留性抗原表位誘導群特異性抗血清:生物資訊學預測

Abstract

豬鐵士古病毒 (Porcine teschovirus, PTV)，為單股正向無封套RNA病毒。PTV目前有13種血清型，19種基因型。在台灣曾於2000年與2004年爆發2次疫情，現今於台灣豬場呈現普遍性地方感染。然而，不同PTV 血清型經常與其他豬病原體在各年齡層共同傳播、共同感染導致各種類似症狀，因而突顯對泛 PTV 診斷工具的需求。本論文旨在利用生物資訊學預測PTV 表位抗原，並發現對各種 PTV 血清型具有群特異反應性的血清試劑；接著利用反向基因方法，以鐵士古病毒為載體構築感染性選殖病毒株，發現將外源基因插入2A位點的重組病毒呈現減毒的現象，提供未來發展減毒載體工具 (含疫苗)的可利用性。我們由生物資訊學預測VP1 的 GH 環上高度保守的“RNNQIPQDF”表位，並且構建帶有泛 DR (PADRE) 和毒素 B 表位的串聯重複的重組蛋白GST–PADRE–(RNNQIPQDF)n–Toxin B作為免疫原，在小鼠體內有效地激發產生針對 PTV非中和或檢測不到的中和抗體。抗血清對所有測試的 PTV 血清型 (PTV 1–7) 都有反應，但對近緣薩佩羅病毒病毒屬和心病毒屬病毒沒有反應，顯示該血清有一定程度的群特異性，這是第一份利用生物資訊學預測PTV 表位發現對各種 PTV 血清型具有廣泛反應性的血清試劑的報告，未來可用於區分自然感染動物和將來發展之不含該表位的次單位疫苗接種的動物 (differentiating infected from vaccinated animals, DIVA)，或在進一步類症鑑別之前篩選 PTV是否存在。此外，構建不含此高度保守表位的病毒載體，期能將泛 PTV 診斷工具應用於未來區別感染動物和不含該表位的次單位疫苗接種的動物。利用反向基因法構築感染性選殖株，將獨特的 XhoI 限制酶切位點引入 2A，以及用 8 組氨酸標記替換 VP1 的 GH 環序列“RNNQIPQDF” 能與PTV原始病毒株鑑別區分。隨後，構築病毒載體攜帶外源基因的可行性研究，將植物螢光蛋白iLOV基因插入PTV結構蛋白基因之間作為標記基因，在不干擾病毒殼蛋白組裝的狀態下復甦重組病毒 (rPTV–iLOV 病毒)，被該重組病毒感染的細胞在螢光顯微鏡下顯現綠光，但由於 iLOV 插入 2A 蛋白酶位點引起的自我切割功能受損降至55.6%，由雙報告基因表達系統評估，顯示有減毒現象。此重組病毒顯示出開發減毒病毒載體疫苗的潛力，幾乎沒有生物安全顧慮，並可作為研究病毒–細胞相互作用的重要工具。 因此，本研究以生物資訊學預測高度保守的抗原表位，開發具有廣泛反應性的血清試劑做為類症診斷之用，尤其是含多血清型之病毒如PTV和流感病毒。本研究並顯示以PTV為平台有助於將來發展減毒病毒載體疫苗及其他應用工具的合理性。

Porcine teschovirus (PTV) is a single–stranded positive non–envelope RNA virus. There are currently 13 serotypes and 19 genotypes of PTV. In recent years, Taiwan have had two outbreaks occurred in years 2000 and 2004, and now it is endemic in pig herds worldwide. However, multiple serotypes of PTV are frequently co–transmitted with other swine pathogens in all age groups and co–infections of different viruses lead to a variety of overlapping or similar symptoms, thus highlighting the need for a pan–PTV diagnostic tool before further differential diagnoses. The aims of this study were to use bioinformatics to predict the PTV highly conserved epitope and generate serological reagents with group–specific reactivity to various PTV serotypes. In addition, PTV was used as the backbone in a reverse genetics system attempted to clone infectious cDNA of PTV, and constructed a PTV–based viral vector that does not contain aforementioned highly conserved epitope but carrying foreign genes, demonstrating the potential of developing a PTV–based vectored vaccines. We predicted the highly conserved 'RNNQIPQDF' epitope on the GH loop of VP1 by bioinformatics, and constructed a recombinant protein GST–PADRE with tandem repeats of pan–DR (PADRE) and toxin B epitopes (RNNQIPQDF)n–Toxin B as an immunogen that efficiently induced non–neutralizing or undetectable neutralizing polyclonal antibodies against PTV in mice. The serum was reactive to all PTV serotypes tested (PTV 1–7), but not reactive to the closely related Sapelovirus and Cardiovirus demonstrating the group–specificity of this serum agent. This is the first report of using bioinformatics to predict a PTV epitope to find serological reagents with broad reactivity to various PTV serotypes, and can be used to distinguish naturally infected from vaccinated animals (DIVA), such as those vaccinated with subunit vaccines that do not contain the aforementioned epitope, and to screen for PTVs prior to differential diagnoses for similar clinical symptoms. In order to construct viral vector without highly conserved epitope for further application of pan–PTV diagnostic tool on DIVA purpose, we used the reverse genetics method to construct infectious clones of PTV cDNA as a backbone, into which a unique XhoI restriction enzyme site was introduced into 2A, and to replace the conserved GH loop sequence 'RNNQIPQDF' of VP1 with an 8–histidine tag that distinguished the rescued virus from the parental PTV successfully. Subsequently, the feasibility of a PTV–based viral vector carrying foreign genes was tested by inserting a plant fluorescent protein gene iLOV between the PTV structural protein genes as a marker. The rescued recombinant PTV (rPTV–iLOV) infected cells were visible as green color under fluorescence microscopy. The insertion of iLOV did not interfere with the assembly of viral capsid proteins, but impaired the self–cleavage of the 2A protease activity to 55.6% (thus attenuation), as assessed by a dual reporter gene expression system. This rescued recombinant PTV shows a potential for developing PTV–based vectored vaccines with few safety concerns and serves as an important tool for the visual study of virus–cell interactions. The results of this study demonstrate the feasibility of predicting highly conserved epitopes by bioinformatics to generate group–specific serological reagent for diagnostic purpose on virus with a multiplicity of serotypes (as in PTV or influenza virus), and it is also feasible to develop PTV–based vector for vaccination and other applications.