構築第二基因型豬流行性下痢病毒之反向遺傳學系統以了解毒力變異相關基因及應用於發展疫苗研究

Abstract

高毒力第二基因型（genogroup 2; G2）豬流行性下痢病毒（Porcine epidemic diarrhea virus; PEDV）感染自2010年末爆發以來，已成為當前最致命且具經濟重要性的豬隻病毒之一。PEDV會造成嘔吐、下痢，並在新生仔豬引起將近百分之百的死亡率，造成全球養豬產業嚴重的經濟損失。過去10年間科學家已慢慢對這個重要豬隻病原有了更進一步的了解，並證實利用活毒減毒疫苗誘發優良的母體免疫是避免新生仔豬遭受PEDV感染的最佳策略。然而，對於PEDV的分子致病機轉、特定病毒基因之功能、或是如何制定合理的疫苗研發方針都仍有待釐清。本篇論文的目標是構築G2 PEDV的反向遺傳系統，並藉此平台研究PEDV的分子減毒機制，以期能應用於研制有效且安全之PEDV減毒活毒疫苗。 本論文第一階段針對一高毒力台灣G2b PEDV分離株（PEDV Pintung 52 passage 5, PEDVPT-P5）及其細胞培養減毒株PEDVPT-P96進行病毒毒力、誘發之宿主免疫保護及序列分析。在五週齡的田間豬模式中，經口給予PEDVPT-P5會誘發豬隻嚴重的水樣下痢與早發且可持續長達26天之糞便排毒。PEDVPT-P96則幾乎不導致任何下痢，其糞便排毒起始時間與排毒高峰的排毒量也都相對較低。經口給予兩種病毒皆可誘發對抗PEDVPT-P5口服攻毒的保護力，唯PEDVPT-P96的保護力較PEDVPT-P5略低。基因組全長分析顯示兩病毒序列長度一致，共有23個核苷酸序列與19個胺基酸序列差異；其中，9個胺基酸變異發生在棘蛋白（Spike; S）基因，特別是S2亞基的區域。上述結果顯示PEDVPT-P96較PEDVPT-P5減毒。儘管有多個胺基酸變異發生於具免疫顯性之棘蛋白，PEDVPT-P96仍保有其免疫原性。 本論文第二部分利用PEDVPT-P96之基因組為骨架以建立PEDV反向遺傳學系統，並以核糖核酸轉染的方法，成功構築了第一個減毒G2 PEDV的感染性選植株。所拯救的重組病毒iPEDVPT-P96與原始之PEDVPT-P96在活體內外皆具有相近之表型，但在五週齡田間豬之攻毒模式中iPEDVPT-P96表現較為減毒。利用次世代定序分析每個核苷酸位點之複雜度，結果顯示iPEDVPT-P96擁有明顯較低之類種（quasispecies）程度，可能與兩病毒表型不完全一致與iPEDVPT-P96較為減毒有關。感染性選殖株iPEDVPT-P96可望將有助研究PEDVPT-P96之減毒決定位，並可用做建立以iPEDVPT-P96作為載體之多價疫苗的研發。 本論文的第三部分旨在研究棘蛋白在PEDVPT 52株減毒過程中的角色。我們建立了四個感染性選植株，分別是高毒力之iPEDVPT-P5、減毒之iPEDVPT-P96，以及將棘蛋白基因互換後之iPEDVPT-P5-96S與iPEDVPT-P96-5S。利用七日齡仔豬進行攻毒試驗，我們發現將強毒iPEDVPT-P5的棘蛋白置換到減毒iPEDVPT-P96的骨架會導致iPEDVPT-P96回毒，造成近乎一致的臨床症狀、糞便排毒以及高死亡率。相反的，同樣的處理卻只讓置換成減毒棘蛋白之iPEDVPT-P5-96S產生部分減毒，並導致較減毒iPEDVPT-P96更為嚴重之組織病變與死亡率。上述結果顯示PEDVPT-P96的減毒主要由棘蛋白基因上的變異造成，但其他基因也在決定毒力上亦扮演一定的角色。 在本研究中，我們成功地建立了G2b PEDV反向遺傳系統，並展示了其用於PEDV減毒機制研究的可行性。本篇論文中所建立之技術與相關發現將有助於PEDV致病機轉之探討，並可實際用於研發有效且安全之PEDV疫苗。

The highly virulent genogroup 2 (G2) porcine epidemic diarrhea virus (PEDV) has become one of the deadliest and economically important swine viruses since its emergence in late 2010. PEDV causes vomiting, diarrhea, and up to 100% mortality rate in neonatal piglets, resulting in tremendous economic losses to the global pig industry. After a decade of efforts aiming to better characterize this important swine pathogen, it is now evident that the desirable maternal immunity induced by live attenuated vaccines (LAV) is essential to provide the neonatal piglets with efficacious protection against PEDV. However, knowledge about the molecular basis of PEDV pathogenesis, specific gene function, and the rational design of PEDV vaccine is yet to be elucidated. Thus, the aim of this dissertation was to develop a reverse genetic system for the G2 PEDV in order to study the molecular mechanism of PEDV attenuation, and thereby propose rational design of safe and effective LAV against PEDV. The first objective was to characterize the genetic, immunoprotective and virulent differences between two autologous Taiwan G2b PEDV strains, the highly virulent PEDV Pintung 52 strain passage 5 (PEDVPT-P5) and its cell culture-adaptive, attenuated counterpart PEDVPT-P96. In the 5-week-old conventional piglet model, oral inoculation with PEDVPT-P5 caused severe watery diarrhea and an early-onset fecal viral shedding that persisted for 26 days. The PEDVPT-P96 induced no to minimal diarrhea and a delayed-onset fecal viral shedding with a lower peak viral load. Both viruses were able to generate protection against challenge with PEDVPT-P5, although the protective efficacy elicited by PEDVPTP96 was slightly lower than that induced by PEDVPT-P5. Sequence analysis of the complete genome of both viruses revealed 23 nucleotide changes and 19 resultant amino acid (a.a.) substitutions with no deletion or insertion. Amongst them, 9 a.a. changes were found in the spike (S) gene, particularly in the S2 subunit. Our data confirmed that PEDVPT-P96 was attenuated compared with PEDVPT-P5 but maintained its immunogenicity despite the presence of several a.a. differences in the immunodominant spike glycoprotein. The second objective was to develop a reverse genetic system of PEDV using PEDVPT-P96 as the backbone. By utilizing the RNA-launched method, we successfully constructed the first infectious cDNA clone of an attenuated G2b PEDV. The rescued recombinant virus (iPEDVPT-P96) exhibited a similar phenotype both in vitro and in vivo, but was further attenuated in the 5-week-old conventional piglet model compared to the parental PEDVPT-P96. Next generation sequencing of both viruses revealed that iPEDVPT-P96 harbored a remarkably decreased level of quasispecies compared to the parental PEDVPT-P96, which may account for the phenotypic differences between the two viruses. The full-length cDNA clone of iPEDVPT-P96 is expected to provide an access to study the attenuation determinants of PEDVPT-P96 and establish an iPEDVPT-P96-based recombinant vector for multivalent vaccine design. Our third objective was to study the role of the S protein in the attenuation process of PEDVPT 52 strain. We generated four infectious cDNA clones, namely the highly virulent iPEDVPT-P5, the attenuated iPEDVPT-P96, and the iPEDVPT-P5-96S and iPEDVPT-P96-5S with substitution of the complete S gene. In 7-day-old piglet model, we found that the replacement of the S gene of PEDVPT-P5 resulted in the complete restoration of the virulence of iPEDVPT-P96 with nearly identical pattern of clinical symptoms, fecal viral shedding, and high mortality rate to the highly virulent iPEDVPT-P5. In contrast, the reciprocal approach only partially attenuated the iPEDVPT-P5 for the iPEDVPT-P5-96S which eventually caused severer histopathological lesions and a higher mortality rate than those induced by the attenuated iPEDVPT-P96. Our data confirmed that the attenuation of the PEDVPT-P96 virus is primarily attributed to mutations in the S gene but other gene(s) might also play a role in determining virulence. To conclude, a reverse genetic system of G2b PEDV was successfully established and its applicability to investigate the attenuating mechanism of PEDV was also demonstrated. The findings and technology obtained herein would facilitate future researches on PEDV pathogenesis as well as safe and effective vaccine development.