豬流行性下痢病毒之病變、診斷試劑開發及抗病毒飼料添加物研究

Porcine Epidemic Diarrhea Virus (PEDV): Studies on the Pathology, Diagnostic Kit Development, and Antiviral Feed Additive

彭茹憶(Ju-Yi Peng)

指導教授 ： 張惠雯

國立臺灣大學/獸醫專業學院/分子暨比較病理生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801238

摘要

自2013年起，臺灣及世界多國出現新型由G2b亞型豬流行性下痢病毒（Porcine epidemic diarrhea virus; PEDV）所引起之大規模的豬流行性下痢(Porcine epidemic diarrhea; PED)，造成重大經濟損失。在此篇論文中，我們探討臺灣PEDV屏東毒株（PEDV-PT 52）之病理學特徵（第二章）、研發監測PEDV特異性抗體之間接型酵素連結免疫吸附法試劑（第三章），同時進行天然芽孢桿菌二次代謝物對抗該毒株（第四章）之效能檢測。
首先，論文第二章探討台灣PEDV-PT毒株之基因序列與病變。在全基因序列中，發現台灣PEDV-PT毒株與北美G2b亞型PEDV毒株高度相近。在病理學研究，我們將PEDV-PT病毒斑純化後以10、103、105 TCID50/mL劑量分別接種於7日齡哺乳豬，再進行顯微組織病理學與免疫組織化學染色偵測PEDV-PT接種之實驗豬及自然感染PEDV-PT之田間豬之病變及腸道組織中病原分佈情形，並與過去文獻中發表之其他PEDV毒株進行比較。結果發現實驗接種PEDV-PT和田間感染PEDV-PT之病理病變相似，然而與已發表之北美G2b亞型PEDV毒株不同。經比較，於PEDV-PT毒株感染豬的十二指腸絨毛及結腸腸上皮無典型PEDV所致之腸絨毛病灶、亦無病毒陽性訊號；在空腸和迴腸之隱窩上皮也無病毒陽性訊號，此與北美G2b亞型PEDV毒株所造成的顯微組織病變有所差異。此研究結果強調比較不同毒株間於腸道組織親和性之差異探討PEDV毒株致病性之重要性。除此之外，藉由實驗證明接種PEDV-PT之豬隻和田間感染PEDV-PT之豬隻的病理病變相似，更說明本實驗動物之攻毒模式與田間感染模式相符，未來該實驗動物之攻毒模式可應用於疫苗及抗PEDV病毒藥物之臨床前驅試驗。
其次，論文第三章中，著重於開發以PEDV全長棘狀蛋白三聚體為抗原之間接型酵素連結免疫吸附法試劑（enzyme linked immune sorbent assay; ELISA）。為表現全長棘狀蛋白三聚體，本實驗構築全長棘狀蛋白基因，並加入T4噬菌體之纖維蛋白基因序列使其成為三聚體結構，後以人類胚胎腎細胞（human embryonic kidney cells ; HEK cell）作為哺乳類細胞之表現系統表現該蛋白質。以病毒感染後之細胞作免疫細胞染色法（immunocytochemical stain; ICC）當作黃金標準，收集138個田間豬隻血清或實驗豬隻血清，將其分為陽性血清ICC組（75個）及陰性血清ICC組（共63個）後，進行PEDV全長棘狀蛋白三聚體之ELISA及商業套組ELISA的臨界值、敏感性特異性及一致性之比較。結果顯示PEDV全長棘狀蛋白三聚體之ELISA臨界值為0.38，作為診斷試劑之敏感性為91%，特異性為92%，且與黃金標準之間有高度的一致性（Kappa=0.82）。然而商業套組試劑ELISA診斷之敏感性僅為31%，特異性為100%，與黃金標準之間僅有低度一致性(Kappa=0.28)。此結果顯示PEDV全長棘狀蛋白三聚體之ELISA為一種具快速且具高信度之血清抗體檢驗試劑，可以作為未來評估疫苗免疫性的重要工具。
最後，論文第四章，主要在評估地衣芽孢桿菌（Bacillus licheniformis）所產生之脂肽生物表面活性劑於活體外與活體內抗PEDV病毒能力及其安全性。實驗結果發現，此生物表面活性劑在150ppm濃度下與感染PEDV之非洲綠猴腎細胞（vero cell）長期培養下具有最顯著之抗病毒效果，其可以顯著抑制病毒之複製與感染之能力，且該作用濃度對於細胞並無細胞毒性。在豬隻試驗結果顯示，四週齡之離乳豬以該生物表面活性劑5kg/ton作為飼料添加劑組具可減緩PEDV-PT攻毒後之臨床症狀並降低病毒排毒量，且在投予豬隻的全身器官之病理檢查並無不良組織反應，也不影響豬隻的每日增重。綜合以上，由地衣芽孢桿菌（Bacillus licheniformis）所產生之脂肽生物表面活性劑，為一可用於降低豬場內豬流行下痢的飼料添加劑。

關鍵字

豬流行性下痢 ； 腸道組織親和性 ； 棘狀蛋白三聚體 ； 間接型酵素連結免疫吸附法試劑 ； 生物表面活性劑 ； 地衣芽孢桿菌 ； 抗病毒藥物評估

並列摘要

The genogroup 2b (G2b) porcine epidemic diarrhea virus (PEDV) that emerged in 2013 has caused devastating disease outbreak and economic losses. In this thesis, we aimed to (1) study the lesion and antigen distributions in piglets challenged with the Taiwan PEDV-Pingtung 52 (PEDV-PT) strain by pathological evaluation (Chapter II); (2) detect PEDV-specific antibodies, and establish an trimeric PEDV spike (S)-based enzyme-linked immunosorbent assay (ELISA) has also been established (Chapter III); (3) evaluate the antiviral activity of biosurfactant derived from the Bacillus licheniformis (B. licheniformis) against PEDV in vitro and in vivo (Chapter IV).
In the first part of the thesis (Chapter II), the full-length genome of the PEDV-PT strain and its intestinal tropism by evaluating the pathological changes in the original PEDV-PT infected field piglet, and 7-day-old conventional piglets orally inoculated with 10, 103, or 105 TCID50/mL of the plaque-purified PEDV-PT-Passage 5 (P5) in were analyzed. Phylogenetic analysis of the full-length genome showed that the Taiwan PEDV-PT strain is closely related to the North American G2b PEDV strains. Some pathological features of the Taiwan PEDV-PT infection, including the absence of lesions and antigen signals in the crypt epithelial cells of the jejunum and ileum and in the villus enterocytes of the duodenum and colon, were different from those of infections by the North American G2b PEDV strains. This difference in the intestinal tropism of the PEDV-PT strain highlights the importance of studying the pathogenicities of different PEDV variants. Moreover, similar distributions of PEDV antigens and lesions in the PEDV-PT infected field piglets and its plaque-purified isolate, PEDV-PT-P5, inoculated piglets indicating that the plaque-purified PEDV-PT-P5 viral stock could facilitate the preclinical evaluation of vaccines and other interventions aimed at preventing the PEDV infection.
In the second part of the thesis (Chapter III), an indirect ELISA coated with PEDV trimeric full-length ectodomain of S protein, generated by incorporation of T4 bacteriophage fibritin trimerization domain and produced by human embryonic kidney cell (HEK), for the detection of PEDV antibody was developed. By using virus-based immunocytochemistry staining (ICC) assay, 138 serum samples divided into the ICC positive group (75 samples) and ICC negative group (63 samples) were used to evaluate and compare the sensitivity and specificity of the trimeric S-protein-based ELISA with a commercial N-protein-based ELISA. The result S showed that the trimeric S-protein-based ELISA had both high sensitivity (91%) and specificity (92%). The agreement between the results of trimeric S-protein-based ELISA and virus-based ICC was statistically significant (Kappa = 0.82). The results demonstrated that the commercial N-protein-based ELISA had a low sensitivity (31%) but a high specificity (100 %) and indicated that the trimeric S-protein-based ELISA has a potential to serve as a reliable tool for detecting PEDV infection, and also a good tool for evaluating the immunogenicity of S-protein-based vaccine.
In the third part of the thesis (Chapter IV), we aimed to evaluate the antiviral activity of the B. licheniformis-derived biosurfactant against PEDV in vitro and its safety and efficacy against PEDV in vivo. In vitro, the results showed while the B. licheniformis -derived biosurfactant exhibited no cell toxicity to Vero cells, co-cultivation of the biosurfactant with PEDV significantly reduced the viral infectivity. In vivo, the result demonstrated that piglets supplemented with B. licheniformis-derived biosurfactant had milder symptom, had lower viral shedding, no significant systemic pathological effect, and no adverse effect on of average daily gain. The results suggest that the biosurfactant derived from B. licheniformis could be a novel candidate as food additive for reducing the manifestation of PED in swine industry.

並列關鍵字

porcine epidemic diarrhea virus ； intestinal tropism ； trimeric spike ； ELISA ； biosurfactant ； Bacillus licheniformis ； antiviral drug

參考文獻

Chapter 1

2. Balasubramanian, B., Li, T., Kim, I.H., 2016. Effects of supplementing growing-finishing pig diets with Bacillus spp. probiotic on growth performance and meat-carcass grade qualitytraits. Revista Brasileira de Zootecnia 45, 93-100.

3. Canning P., Ruston C., Mason D., Bate J., Skoland D., Davenport J., Gaul S., Wang C., Chen Q., Zhang J., Karriker L., 2017. Effect of direct-fed microbial Bacillus subtilis C-3102 on enteric health in nursery pigs after challenge with porcine epidemic diarrhea virus. J Swine Healt and Product 25, 129-137.

4. Chang S.H., Bae J.L., Kang T.J., Kim J., Chung G.H., Lim C.W., 2002. Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus. Molecules and Cells 14, 295-299.

5. Chen, Q., Gauger, P.C., Stafne, M.R., Thomas, J.T., Madson, D.M., Huang, H., Zheng, Y., Li, G., Zhang, J., 2016. Pathogenesis comparison between the United States porcine epidemic diarrhoea virus prototype and S-INDEL-variant strains in conventional neonatal piglets. J Gen Virol 97, 1107-1121.