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| 研究生： |
克雷杰 Kraijak Kaewprom |
|---|---|
| 論文名稱： |
台灣豬生殖與呼吸綜合症研究：基因演化、第一型病毒之鑑定
和草藥萃取物之抗病毒活性 Porcine Reproductive and Respiratory Syndrome (PRRS) Research on the Genetic Evolution, Identification of a Type 1 Virus and Antiviral Activity of Herb Extracts in Taiwan |
| 指導教授： |
林昭男
Chao-Nan Lin 邱明堂 Ming-Tang Chiou |
| 學位類別： |
博士 Doctor |
| 系所名稱： |
獸醫學院 - 獸醫學系所 Department of Veterinary Medicine |
| 畢業學年度： | 109 |
| 語文別： | 英文 |
| 論文頁數： | 107 |
| 中文關鍵詞： | 豬生殖與呼吸綜合症 、第五開放讀碼區 、演化 、中藥萃取物 、螢光定量聚合酵素鏈鎖反應 、台灣 |
| 外文關鍵詞： | PRRSV, ORF5, Evolution, Herbal Extracts, qPCR, Taiwan |
| DOI URL： | http://doi.org/10.6346/NPUST202100020 |
| 相關次數： | 點閱：25 下載：3 |
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本次實驗首先探討從2002年至2014年間，台灣七間不同豬場的豬生殖與呼吸綜合症病毒（PRRSV）的第五開放讀碼區（ORF5）進行基因序列演化分析調查，來分析疾病動力學在不同等級場內的感染模式。首先從七座（A、E、F、H、L、S與U）不同豬場，蒐集30份檢體，進行核酸序列分析，結果顯示所有序列彼此相似度皆低於97%，並被歸類在不同集群，其中來自同一牧場的最後一個分離株，被認為是外來菌株。本次序列分析研究結果與不同第二型PRRSV做比較，與VR-2332病毒株相比，相似度為84.9-88.4%；與Lelystad病毒株相比，相似度為62.1-65.3%；與MD001病毒株相比，相似度為85.9-89.6%，這些第二型PRRSV皆為各自獨立演化的群集。其中從A牧場蒐集的兩條PRRSV序列，雖時間相隔十年，其基因相似度為95.5%，被歸屬在相同群集，推測為相同來源的疫情爆發。剩下的六座牧場，在相同疫情爆發時間，至少皆有一外來病毒株。我們得到結論為外來PRRSV病毒株在台灣不同豬場的散佈與入侵模式皆相似。本次研究接著探討在台灣是否存在第一型豬生殖與呼吸綜合症病毒（type 1 PRRSV），我們調查先前疑似的一個案例，並進行基因、親源分析，命名為PRRSV/NPUST-2789-3W-2/TW/2018 (NPUST2789)，其親緣關係與歐洲型PRRSV疫苗株較為接近。本病毒株是在台灣中部某豬場於2018年十月至十一月期間爆發，而後被成功分離。本場懷孕母株有出現流、死產，小豬則是有呼吸道症狀。實驗室檢驗發現母豬及小豬的血清皆有PRRSV 1病毒核酸存在，透過全基因定序後發現與歐洲疫苗序列相似度高達98.2%。本篇研究為首例在台灣爆發PRRSV 1疫情，且與歐洲疫苗株有相同演化源。本篇最後研究主軸在評估藥草萃取物是否在臨床上能提供抗PRRSV感染能力。本研究從42種藥草萃取物中，尋找能抵抗兩株本土PRRSV分離株感染Marc-145細胞。根據細胞存活率試驗結果顯示，高濃度的植物萃取物與其他相比，在Marc-145細胞之細胞毒性少10%。抗病毒試驗結果顯示，Thymus vulgaris
與Nepeta cataria皆能在活體外感染試驗中，有顯著低的病毒量，其抗病毒機制仍需再被深入探討。本研究結果顯示試驗前、後階段，Thymus vulgaris
與Nepeta cataria皆能降低PRRSV所造成10%的細胞毒性。綜合所有結果，Thymus vulgaris與Nepeta cataria皆具有發展疾病預防及PRRSV感染治療的潛能。
The first experiment focused on investigating genetic evolution of PRRSV based on sequencing ORF5 gene in 7 from Taiwanese pig farms during 2002 to 2014 to analyze the disease spread within herds for better understanding the dynamics of PRRSV at the farm level. Herein, thirty ORF5 sequences from 7 Taiwanese pig farms (A, E, F, H, L, S, and U) were collected. Nucleotide sequences showed an identity of less than 97% and belonging to different clusters than last isolates from the same farm were considered to represent exotic strains. The results showed that the identities among these isolates were 84.9-88.4% (VR-2332), 62.1-65.3% (Lelystad) and 85.9-89.6% (MD001) to PRRSV type II. All of these studied strains belonged to PRRSV type II and evolved separately. Two nucleotide sequences of PRRSV strains from Farm A collected 10 years apart showed 95.5% of the identity but belonged to the same cluster were considered to be the same strain endemic to the farm. In the rest of 6 farms, at least 1 exotic strain was detected during that period. We concluded that in the area, the spread and new invasions of exotic PRRSV strains at the farm level are common events in Taiwan. The second experiment focused on the identification of an unidentified type 1 PRRSV (PRRSV 1) in Taiwan. We investigated the previously unidentified PRRSV 1 outbreak in Taiwan. We reported a genetic and phylogenetic analysis of the PRRSV 1 isolate, designated PRRSV/NPUST-2789-3W-2/TW/2018 (NPUST2789), which was genetically closely related to that of a European PRRSV vaccine strain. Then it was isolated during October to November 2018, in a pig herd in central Taiwan. Pregnant sows had abortions and stillbirths, and piglets suffered from respiratory disorders. Laboratory tests identified the presence of PRRSV 1 in serum from sows and suckling piglets in this event. The complete genome of the identified PRRSV 1 strain was genetically closely related to that of a European PRRSV vaccine strain (98.2%). This is the first report on suggestion on outbreak in Taiwan of a PRRSV 1 strain that shared a common evolutionary ancestor with the European PRRSV vaccine strain. The third experiment focused on that medicinal herb extracts may provide clinically protective against PRRSV infection. Fourty-two natural herb extracts were screened for their antiviral activity against two local PRRSV strains in MARC-145 cells. Based on the results obtained from the cell viability tests, those with the highest concentration showing less than 10% cytotoxicity in MARC-145 cells were chosen for each for the plant extracts. The antiviral activity revealed that both Thymus vulgaris and Nepeta cataria extracts were able to significantly reduce the PRRSV load in vitro. The anti-PRRSV mechanisms were further investigated. The results indicated that the anti-PRRSV activity of < 10% cell cytotoxicity both T. vulgaris and N. cataria extracts of both pre-entry and post-entry steps. These results suggested that both T. vulgaris and N. cataria extracts had therapeutic potential in the prophylaxis and treatment of PRRSV infection.
中文摘要............................................................................................................ I
Abstract ........................................................................................................... III
Acknowledgements ........................................................................................... V
Table of Contents ............................................................................................ VI
List of Tables ................................................................................................... IX
List of Figures ................................................................................................... X
Chapter 1. Introduction ...................................................................................... 1
1.1 Introduction ......................................................................................... 1
1.2 Objectives ............................................................................................ 6
Chapet 2. Literature Review............................................................................... 7
2.1 Porcine Reproductive and Respiratory Syndrome Disease (PRRS)... 7
2.1.1 Causative Agent ........................................................................... 7
2.1.2 Replication and Transcription of PRRSV.................................... 9
2.1.3 Virus Assembly and Budding .................................................... 11
2.1.4 PRRSV Cell Tropism................................................................. 12
2.1.5 Pathogenesis............................................................................... 13
2.1.6 Clinical Signs and Pathology ..................................................... 17
2.1.7 Immune Response ...................................................................... 18
2.1.8 Genetic Diversity ....................................................................... 19
2.1.9 Diagnostic Tools ........................................................................ 21
2.1.10 Control, Prevention and Treatment.......................................... 24
2.2 Medicinal Plants................................................................................ 25
2.2.1 Thymus Vulgaris........................................................................ 26
2.2.2 Nepeta Cataria............................................................................ 31
Chapter 3. Genetic Evolution of Porcine Reproductive and Respiratory
Syndrome Virus Based on ORF5 on 7 Taiwanese Pig Farms ..........35
3.1 Purpose of the Study ......................................................................... 35
3.2 Materials and Methods...................................................................... 35
3.2.1 Specimen Collection .................................................................. 35
3.2.2 ORF5 Amplification and Sequencing........................................ 35
3.2.3 Phylogenetic Analysis................................................................ 36
3.2.4 Amino Acid Sequence Analysis ................................................ 36
3.3 Results ............................................................................................... 36
3.3.1 Phylogenetic Analysis................................................................ 36
3.3.2 ORF5 Nucleotide Sequences Analysis ...................................... 37
3.3.3 ORF5 Nucleotide Sequences Analysis During a 5-7 Years
Sampling Period .................................................................................. 37
3.3.4 ORF5 Nucleotide Sequences Analysis During a 2-3 Years
Sampling Period .................................................................................. 38
3.3.5 Amino Acid Variation in GP5 ................................................... 38
3.4 Discussion and Conclusion ............................................................... 43
Chapter 4. Identification of an Unidentified PRRSV Type I Virus in Taiwan 47
4.1 Purpose of the Study ......................................................................... 47
4.2 Materials and Methods...................................................................... 47
4.2.1 Clinical Cases............................................................................. 47
4.2.2 Sample Preparation and Quantification of PRRSV RNA.......... 47
4.2.3 Serology ..................................................................................... 48
4.2.4 Virus Isolation and Whole Genome Sequencing of PRRSV..... 48
4.2.5 Open Reading Frame 5 Amplification and Sequencing ............ 48
4.2.6 Sequence and Phylogenetic Analysis ........................................ 49
4.3 Results ............................................................................................... 49
4.3.1 Emergence of PRRSV 1............................................................. 49
4.3.2 Phylogenetic Analysis of Whole Genome Sequences ............... 50
4.3.3. Phylogenetic, Amino Acid and Nucleotide Sequence Analysis
of ORF5............................................................................................... 50
4.4 Discussion and Conclusion ............................................................... 59
Chapter 5. Antiviral Activity of Medicinal Herb Extracts on Field PRRSV
Strain ................................................................................................. 61
5.1 Purpose of the Study ......................................................................... 61
5.2 Materials and Methods...................................................................... 61
5.2.1 Preparation and Purification of Natural Plant Hydrosols .......... 61
5.2.2 Cell and Viruses ......................................................................... 61
5.2.3 Cell Viability.............................................................................. 62
5.2.4 Screening of Antiviral Activity.................................................. 62
5.2.5 Pre-entry Assay .......................................................................... 63
5.2.6 Post-entry Assay ........................................................................ 64
5.2.7 Real-time PCR and Indirect Immunofluorescence Assay (IFA)64
5.2.8 Statistical Analysis ..................................................................... 64
5.3 Results ............................................................................................... 65
5.3.1 Cell Viability and Cytotoxicity.................................................. 65
5.3.2 Screening of 42 Natural Plant Hydrosols for Antiviral Activity65
5.3.3 Anti-PRRSV Mechanisms of T. Vulgaris and N. Cataria
Hydrosols ............................................................................................ 66
5.4 Discussion and Conclusion ............................................................... 77
Chapter 6. General Discussion and Conclusion ...............................................80
7. References ....................................................................................................83
Bio-sketch of Author ......................................................................................102
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