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Updated Phylogenetic Analysis of Spike Gene of Porcine Epidemic Diarrhea Virus in Taiwan and the Strategy of Vaccine Development for Inducing Mucosal Immunity in Pigs
porcine epidemic diarrhea virus (PEDV),phylogenetic analysis,recombination,CC chemokines (CCL27, CCL28 and CCL25),mucosal immunity,
|Publication Year :||2019|
|Abstract:||自2013年末開始，臺灣爆發由新型G2b亞型豬流行性下痢病毒（porcine epidemic diarrhea virus; PEDV）所引起之豬流行性下痢（porcine epidemic diarrhea; PED）。此疾病以急性嚴重下痢、劇烈嘔吐、脫水甚至死亡為特徵，重創台灣養豬產業。自2015年後，此新型G2b PEDV轉而成為週期性爆發的疾病。為了暸解於2016以後臺灣PEDV基因序列之轉變，在本論文研究中，首先（第二章），將討論臺灣於2016到2018期間，PEDV棘突基因（spike; S gene）中的序列變化，並與過去臺灣及全世界PEDV病毒株進行序列分析比對；接著使用次世代定序（next generation sequencing; NGS）針對可能重組的PEDV病毒株進一步分析。此外，由於成功誘導黏膜免疫一直以來為研發PEDV疫苗之最大瓶頸，本論文第三章則為構築及表現豬源趨化激素（CC chemokines）包含CCL27、CCL28及CCL25蛋白作為誘發黏膜免疫之佐劑，並以離乳豬為模式評估豬源趨化激素CCL27、CCL28及CCL25是否能成功誘發黏膜免疫及保護豬隻免於PED之致害，以發展能刺激黏膜免疫反應為導向之新型疫苗。
在第二章節中，本研究在2016到2018期間，總共收集31株源於28個不同的豬場PED確診之腸道或糞便之PEDV病毒株。經由棘突蛋白基因全長定序結果顯示，除了TW/Yunlin550/2018此序列外，臺灣大部分PEDV序列仍皆與全世界G2b PEDV較為相似；並於棘突蛋白的中和抗體決定位中，發現臺灣近年PEDV序列相較於過去G2b PEDV序列出現較多變異的胺基酸位點。另外，經由棘突蛋白及次世代全基因全長定序更確認TW/Yunlin550/2018病毒株為一新型基因分群G1c，其可能為G2b與G1a重組過後的病毒株。
The newly emerging variant of group 2b (G2b) porcine epidemic diarrhea virus (PEDV) leading to porcine epidemic diarrhea (PED) with characteristic of acute severe diarrhea and vomiting, dehydration or even death has devastated the swine industry in Taiwan since late 2013. After 2015, the novel G2b PEDV variant further transformed into a cyclical outbreak. To understand the diversity of PEDVs circulating in Taiwan after 2016, phylogenetic analysis of genetic sequence of the S gene of PEDVs collected during 2016-2018 in Taiwan were compared with the historic and global PEDV strains (Chapter II). The next generation sequencing (NGS) was further performed for the analysis of a potential recombinant Taiwan PEDV variant. In addition, the successful induction of mucosal immunity has always been a bottleneck for the development of vaccines by route of intramuscular (IM) administration. In Chapter III, three porcine CCL proteins (CCL27, CCL28, and CCL25) derived from mammalian cell expression system were constructed and applied as mucosal immune adjuvants with an inactivated PEDV (iPEDV) as immunogen by IM immunization. The immunogenicity and protective efficiency of the iPEDV adjuvanted with different combinations of CC chemokines (CCL27, CCL28 and/or CCL25) were evaluated in 5-week-old pigs by IM immunization to develop the novel vaccine strategy for inducing mucosal immune responses.
In Chapter II, a total of 31 intestinal and/or fecal specimens from 1-day to 6-week old piglets or sows with watery diarrhea confirmed to be PEDV-positive by quantitative reverse transcription polymerase chain reaction (RT-qPCR) from 28 different herds in Taiwan during 2016-2018 were sequenced and analyzed. The phylogenetic analysis of the S gene of PEDV revealed that the majority (28/31) of Taiwan new PEDV strains, except for the TW/Yunlin550/2018 strain, were closely related to the global G2b PEDV strains with unique deletions and insertions scattered in the neutralizing epitope regions of the S protein as compared to the historic G2b PEDV strains. Moreover, the TW/Yunlin550/2018 strain was proposed a recombinant between a G2b strain and a circulating wild-type PEDV G1 strain based on the results of sequencing of S gene and NGS of the whole PEDV genome.
In Chapter III, three porcine CCL proteins (CCL27, CCL28, and CCL25) derived from the mammalian cell expression system were stably expressed and applied as vaccine adjuvants. In the experiment of post-weaning pigs, our data showed that IM co-administration of iPEDV with the above mentioned chemokines as adjuvants for two times at a two-week interval could induce higher levels of systemic PEDV-specific immunoglobulin (Ig) G, mucosal PEDV-specific IgA, and viral neutralizing antibody titer; and better substantial protection against PEDV challenge than those administrated without CC chemokines and in control pigs. The present study verified that IM immunization with different combinations of porcine CC chemokines could trigger their unique immune trafficking abilities to elevate the antigen-specific systemic and mucosal immune responses simultaneously. These porcine CC chemokines can be used in the vaccine development for not only PEDV but also other mucosal transmissible swine diseases.
|Appears in Collections:||分子暨比較病理生物學研究所|
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