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研究生:林育如
研究生(外文):Yu-Ju Lin
論文名稱:台灣本土型與外來型豬瘟病毒遺傳與致病力之比較
論文名稱(外文):Comparison of genetics and pathogenicity between the historical and new invaded classical swine fever viruses in Taiwan
指導教授:簡茂盛簡茂盛引用關係
指導教授(外文):Maw-Sheng Chien
學位類別:博士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
畢業學年度:96
語文別:英文
論文頁數:81
中文關鍵詞:豬瘟病毒本土型外來型致病力
外文關鍵詞:classical swine feverhistorical straininvaded strainpathogenicity
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豬瘟 (classical swine fever; CSF) 為豬隻之高度傳染性及高致死性的病毒性疾病,本病的爆發往往造成重大的經濟損失。台灣田間分離的豬瘟病毒主要分成二型,本土型(3.4基因亞型)與外來型(2.1基因亞型)。本土型的豬瘟病毒分佈,只限於日本南方到台灣地區,且爆發病例只出現在1996年前。
為瞭解二型病毒之遺傳特性,因此本研究針對本土型豬瘟病毒(3.4基因亞型),以參考病毒株94.4/IL/94/TWN進行全長定序。基因體全長具有12,296個核苷酸,可轉譯成3,898個氨基酸,基因體前後包含有5端非轉譯區 (5′UTR) 372核苷酸及3端非轉譯區 (3′UTR) 227核苷酸。台灣地區的3.4基因亞型與同一基因亞型的日本神奈川株(1974年)及沖繩株(1986年)核苷酸的相似度維持在94.2~97.5% ; 然而與1.1基因亞型的強毒株 (ALD/64/Jap)及2.1基因亞型的病毒株 (TD/96/TWN) 的相似度只有72.5~80.8%。以NS5B核苷酸相對應位置11,157∼11,565的基因片段進行演化分析,可將豬瘟病毒區分成三個基因亞型。94.4/IL/94/TWN參考株為第一株完成全長定序的3.4基因亞型病毒株。
直到目前為止,尚未找到與毒力有關的基因決定區。本研究推論台灣田間病毒基因型別之間的轉換,可能是因為不同基因型別病毒的增殖速率不同,或是病毒毒力的差異所造成。為瞭解病毒在細胞培養中的增殖情形,本研究選取了豬腎臟細胞株、初代豬睪丸細胞及豬肺泡巨噬細胞進行病毒增殖速率分析。結果顯現94.4/IL/94/TWN及PT/96/TWN二株病毒在豬腎臟細胞株,初代豬睪丸細胞及豬肺泡巨噬細胞有相似的增殖速率。然而在以豬進行的活體增殖實驗中,PT/96/TWN在感染晚期時毒血及各組織臟器則具有較快的增殖速率。
為了進一步瞭解本土型與外來型豬瘟病毒的毒力及表徵,因此本研究設計出一病理積分表,包含肉眼及組織病理病變二部分,嘗試將病變的嚴重度及病程的長短進行區分及量化。此外為瞭解病毒毒力的差異,選擇無毒力的兔化豬瘟疫苗病毒株(LPC)及高毒力的標準強毒株ALD作為豬瘟病毒毒力的指標,進行比較。實驗結果發現94.4/IL/94/TWN及PT/96/TWN病毒株在豬隻感染後的任何時間點的病理積分均具有顯著的差異。然而以LPC及ALD分別作為最低與最高的參考基準,因此本研究將94.4/IL/94/TWN及PT/96/TWN病毒株歸屬於中間毒力的病毒。
本研究認為病毒的毒力是可以利用病變的嚴重度及病毒在活體中的增殖速率作為評估的依據。在本實驗中,本研究雖然無法明確的解釋界定病毒的毒力,但是本研究嘗試建立應用病理積分的方式評估不同型別之豬瘟病毒、毒力及表徵的差異。
Classical swine fever, formerly referred to as hog cholera, is a highly contagious and fatal disease of pigs. Outbreaks of this disease caused significant economic losses in the pig industry all over the world. Classical swine fever viruses in Taiwan have been classified into two subgroups (3.4 and 2.1). Outbreaks caused by 3.4 viruses were reported in Taiwan prior to 1996 and mainly distributed in the geographic area ranging from southern Japan to Taiwan.
To identify genetic differences, the complete sequence of 94.4/IL/94/TWN, one of the reference strains of subgroup 3.4, was determined. The genome contains 12,296 nucleotides, encoding 3,898 amino acids flanked by a 372-nt region at the 5′ untranslated region (UTR) and a 227-nt region at the 3′-UTR. Similarities of nucleotides among 3.4 viruses isolated from Taiwan and Japan (Kanagawa/74; Okinawa/86) maintained in 94.2 to 97.5%; however, comparing to subgroup 1.1 (ALD/64/Jap) and 2.1 (TD/96/TWN) only showed about 72.5 to 80.8% similarity, respectively. Phylogenetic analysis based on positioning from 11,157 to 11, 565 nt (NS5B) revealed that CSFVs were divided into three major lineages similar to previous studies using E2 or NS5B regions. Strain 94.4/IL/94/TWN is the first completely genome sequenced in subgroup 3.4 viruses.
Until now, however, no genetic determinant of virulence is defined. We speculate that the dramatic change(s) of genotypes in field outbreaks may be caused by the difference(s) in viral replication rate or virulence. Kinetics of viral replication with different genotypes were evaluated in vitro and in vivo. The results indicated that the invaded strain had a higher virus titer at the later stage of infection in vivo; however, both strains replicated well and display similar kinetics in PK-15, PAM and ST cells.
In order to clarify the virulence in phenotypes between historical and invaded strains, a new pathological (gross and histopathology) score was introduced. The avirulent vaccine strain (lapinized virus strain, LPC) and highly virulent strain (ALD) were used as disease indicators. The pathological lesions caused by both strains did present significant differences at various time points postinfections. Even though it is distinguishable for their virulent ability, by comparing to LPC and ALD strains, both 94.4/IL/94/TWN and PT/96/TWN viruses should be defined as moderate virulent strains.
Based on the results, the virulence of CSFV might be determined by its replication ability in animals. In this study, we did not specifically intend to explain viral virulence; whereas, we established a new pathological score to evaluate the virulence differences and phenotypes in pigs infected by various virus strains.
Abstract in Chinese --------------------------------------------------------------------- i
Abstract -------------------------------------------------------------------------------- iii
Contents -------------------------------------------------------------------------------- v
List of Tables ---------------------------------------------------------------------------- viii
List of Figures --------------------------------------------------------------------------- ix
Chapter 1 Introduction ----------------------------------------------------------------- 1
Chapter 2 Literature Review ---------------------------------------------------------- 2
2.1 Historical background ----------------------------------------------------- 2
2.2 Etiology ------------------------------------------------------------------ 3
2.2.1 Classification --------------------------------------------------- 3
2.2.2 Genome organization and replication ----------------------- 3
2.2.2.1 Genome -------------------------------------------------- 3
2.2.2.2 Structural protein --------------------------------------- 3
2.2.2.3 Non-structural protein ---------------------------------- 5
2.2.2.4 5′- and 3′-UTR ------------------------------------------- 5
2.3 Genetic typing of CSFV and epidemiology ------------------------- 7
2.4 Biological properties --------------------------------------------------- 7
2.4.1 Virulence ------------------------------------------------------- 8
2.4.2 Clinical signs --------------------------------------------------- 8
2.4.3 Pathogenesis --------------------------------------------------- 9
2.5 Diagnosis ---------------------------------------------------------------- 10
2.5.1 Direct detection of CSFV ------------------------------------ 11
2.5.1.1 Virus isolation ------------------------------------------- 11
2.5.1.2 Detection of CSFV antigen ---------------------------- 11
2.5.1.3 Detection of CSFV nucleic acid ---------------------- 11
2.5.2 Detection of antibodies against CSFV ---------------------- 12
2.6 Prevention and control ------------------------------------------------- 12
2.6.1 Transmission --------------------------------------------------- 12
2.6.2 Control ---------------------------------------------------------- 12
Chapter 3 Complete sequence of a subgroup 3.4 strain of classical swine fever virus from Taiwan ----------------------------------------------------------
15
3.1 Abstract ------------------------------------------------------------------ 15
3.2 Introduction ------------------------------------------------------------- 15
3.3 Materials and methods------------------------------------------------- 17
3.4 Results ------------------------------------------------------------------- 19
3.5 Discussion --------------------------------------------------------------- 22
Chapter 4 Kinetics of viral replication in various classical swine fever virus strains in vitro and in vivo -------------------------------------------------
25
4.1 Abstract ------------------------------------------------------------------ 25
4.2 Introduction ------------------------------------------------------------- 25
4.3 Materials and methods ------------------------------------------------- 26
4.4 Results -------------------------------------------------------------------- 29
4.5 Discussion --------------------------------------------------------------- 33
Chapter 5 Characterization of the virulence of historical (subgroup 3.4) and new invaded (subgroup 2.1) classical swine fever virus ---------------
35
5.1 Abstract ----------------------------------------------------------------
5.2 Introduction -------------------------------------------------------------- 35
35
5.3 Materials and methods ------------------------------------------------- 37
5.4 Results -------------------------------------------------------------------- 39
5.5 Discussion --------------------------------------------------------------- 42
Chapter 6 Conclusions ------------------------------------------------------------------ 45
References -------------------------------------------------------------------------------- 64
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