台灣豬瘟病毒分子流行病學之研究

Abstract

豬瘟是由豬瘟病毒引起之高接觸性傳染病，親緣樹分析有助於追蹤豬瘟病毒發生的地理起源並了解病毒基因型別的分佈。將1989至2007年台灣分離到的167株豬瘟病毒，針對Erns及E2封套醣蛋白基因以RT-PCR增幅此兩區間並進行核酸定序及親緣樹分析。雖然兩者出現類似的樹形圖，但Erns比E2具有更好的區別效果。親緣樹分析結果顯示，124個田間分離株屬於2.1及2.2亞群，此兩亞群都屬於外來型病毒株，其餘的43個田間分離株則屬於3.4亞群的本土型病毒株。由於2.1亞群病毒株在Erns親緣樹分析時可進一步區分成信賴值(bootstrap values)高達98%及85%的兩個群組(clusters)，顯示此兩群組間病毒的核酸序列有明顯差異，因此我們認為2.1亞群應再進一步區分為2.1a及2.1b。其中2.1a 亞群病毒株最早於1994年入侵台灣，1995年即爆發流行，之後成為田間優勢族群至今。然而3.4亞群病毒株盛行於1994年以前，但自1996年以後就無法從田間分離到此型病毒。過去近二十年間，我們看到台灣田間流行的豬瘟病毒從3.4亞群轉變成2.1a 亞群，這基因型轉變並非由本土型病毒株的基因突變所造成。從基因序列分析結果發現，2.1a亞群病毒與德國分離株Paderborn及寮國分離株L119最相近，然而2.1b 亞群與中國廣西省分離株最為相近。另針對完整的封套醣蛋白(Erns- E2)核酸序列(2,385 bp)以親緣樹分析27 個豬瘟病毒之核酸序列，發現此區間相較於全長的開放讀碼區(Open reading frame)核酸序列(11,691 bp)具有更好的區別效果，可以將具親緣關係之ALD病毒及GPE- 疫苗毒群集一起(cluster together)。兩株外來型病毒(基因型2.1a 及 2.1b)被選來評估LPC兔化豬瘟疫苗對於豬隻的保護效力試驗，SPF豬以不同劑量(1, 1/10及1/100劑量)之LPC疫苗免疫，14天後分別以基因型2.1a 及 2.1b外來型豬瘟病毒進行攻毒試驗。結果顯示，目前台灣使用的LPC兔化豬瘟疫苗可以完全保護這兩株外來型豬瘟病毒的攻擊。豬瘟活毒減毒疫苗免疫小豬後疫苗毒會持續存在豬隻體內一段時間，一般實驗室常用的RT-PCR檢測方法無法區別野外毒及疫苗毒，需將PCR產物進行核酸定序以排除被檢病例中來自疫苗毒之干擾。本論文基於兔化豬瘟疫苗毒核酸序列中有一段T-rich插入之特性，發展單一步驟RT-PCR檢測方法。使用單步驟RT-PCR或巣式RT-PCR增幅，經傳統洋菜膠或毛細管電泳後，可同時檢測及區別臨床檢體中的豬瘟野外毒及兔化豬瘟疫苗毒。本方法至少可應用於LPC、HCLV及C-strain等三種兔化豬瘟疫苗毒，對於豬瘟野外毒檢測的敏感度RT-PCR及巣式RT-PCR分別為6.3及0.63 TCID50/ml。前人報告指出，兔化豬瘟疫苗毒核酸序列3' 端未轉譯區有一段12至13個T-rich插入之特性，然而我們發現LPC/PRK及LPC/TS兩株疫苗毒T-rich插入片段長度分別多達42及36個核苷酸，這些不同大小的T-rich插入片段增加RT-PCR產物的大小，可當做很好的基因標記藉以快速區別豬瘟野外毒及不同兔化豬瘟疫苗毒株。本論文進一步開發DNA 晶片檢測方法以達到同步檢測、分型及區別豬瘟野外毒及兔化豬瘟疫苗毒之目標。豬瘟病毒特異性引子及探針設計在病毒基因3' 端未轉譯區，先利用生物素標示引子(biotin-labeled primer)進行單步驟RT-PCR增幅，隨後將增幅產物與固定在塑膠晶片上的DNA探針進行雜合反應。利用DNA晶片方法不僅可將豬瘟病毒區分成三種主要基因型，亦可同時區別豬瘟野外毒及兔化豬瘟疫苗毒。RT-PCR及DNA晶片檢測之敏感度分別為10 及1 TCID50/ml，DNA晶片檢測方法之敏感度較RT-PCR方法高約10倍。RT-PCR結合DNA探針雜合技術可提供一高敏感性的檢測工具，適用於臨床病例中豬瘟病毒之檢測、分型及區別野外毒及疫苗毒。

Classical swine fever (CSF) is a highly contagious viral disease of swine caused by classical swine fever virus (CSFV). Phylogenetic analysis of CSFV field isolates are useful to trace the geographic origins of the disease and to understand the distribution of CSFV genotypes. Two envelope glycoprotein (Erns and E2) regions of CSFV were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced directly from 167 specimens collected between 1989 and 2007 in Taiwan. Phylogenetic analysis of the two regions revealed a similar tree topology and, furthermore, the Erns region provided better discrimination of CSFV genotypes than the E2 region. Of the 167 isolates collected, 124 were clustered within subgroups 2.1 and 2.2, which were considered to be potential exotic strains, whereas the remaining 43 isolates were clustered within subgroup 3.4, which is considered to contain the historical strains. Since the subgroup 2.1 could be further separated into two different clusters with high bootstrap values of 98% and 85% in the Erns tree, we proposed that subgroup 2.1 should be further classified as 2.1a and 2.1b. The subgroup 2.1a viruses were introduced to Taiwan in 1994 and caused CSF outbreaks in 1995, and then predominated in the field onwards. However, the subgroup 3.4 viruses were prevalent in Taiwan prior to 1996 and seemed to disappear from the field since it could not be isolated from the field thereafter. We have observed a dramatic switch in genotype from subgroup 3.4 to 2.1a in the last two decades. The subgroup 2.1a isolates are closely related to the Paderborn and Lao isolates, whereas 2.1b isolates have a close relationship to the Chinese Guangxi isolates. The phylogenetic tree of 27 CSFV sequences based on the complete envelope glycoprotein gene (containing 2,385 bp) displayed better resolution than that based on the complete open reading frame gene (containing 11,691 bp). The ALD strain showed cluster together with the GPE- vaccine virus only in the complete envelope glycoprotein gene tree. Two exotic viruses with the genotypes 2.1a and 2.1b were selected as challenging candidates to evaluate the protective efficacy of the LPC vaccine. SPF pigs were vaccinated with various dosages (1, 1/10, and 1/100 doses) of LPC vaccine and challenged with a CSFV exotic strains, either from genotype 2.1a or from genotype 2.1b. The results demonstrated that the LPC vaccine that is currently used in Taiwan could provide full protection against these two exotic CSFVs. Live attenuated vaccine strains of CSFV can persist in the tonsils and lymph nodes of piglets for a long period of time after immunization. Routinely, RT-PCR followed by DNA sequencing has been the method used to detect CSFV and excludes the interference of vaccine viruses in field cases. Herein, a simple one-step RT-PCR method was developed, based on T-rich insertions in the viral genome, for simultaneous detection and differentiation of wild-type and vaccine strains of CSFV. The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. Using a one-step RT-PCR or a semi-nested RT-PCR followed by agarose gel or multi-capillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least three attenuated lapinized vaccine strains, LPC (lapinized Philippines Coronel), HCLV (hog cholera lapinized virus), and C (Chinese)-strain. The detection limit for the wild-type virus was 6.3 TCID50 (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID50/ml for semi-nested RT-PCR. In previous studies, notable T-rich insertions of 12–13 nucleotides (nts) were found in the 3' NTR of the genome of CSFV lapinized vaccine strains. However, this study discovered that two T-rich insertions of 42 and 36 nts are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nts increases the size of PCR fragments, which are thus simple genetic markers for the rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV. This study also developed a DNA chip to enable simultaneous detection, genotyping, and differentiation between wild-type and vaccine-type CSFV. One-step RT-PCR amplification was performed with biotin-labeled primers, followed by hybridization to the DNA probe immobilized on the plastic chips. The DNA chip can not only accurately differentiate between the three major genotypes of CSFV, but can also discriminate between the wild-type and vaccine-type CSFV. The limit of detection for the wild-type virus was 10 TCID50/ml for RT-PCR and 1 TCID50/ml for the DNA chips. The sensitivity of the visual DNA chip was 10 times higher than that of the RT-PCR, as confirmed by agarose gel. The RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping CSFV and discriminating between vaccine and wild-type CSFV in clinical samples.