臺灣豬環狀病毒分子流行病學之研究

Molecular Epidemiological Studies of Porcine Circovirus in Taiwan

王羣(Chun Wang)

指導教授 ： 鄭謙仁

共同指導教授 ： 賴秀穗

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.10101

摘要

第二型豬環狀病毒（porcine circovirus type 2, PCV2）為豬隻主要的病毒性病原，其已在世界各地包括台灣的養豬場造成重大的經濟損失。PCV2被認為是引起豬隻離乳後多系統消耗性症候群 (postweaning multisystemic wasting syndrome, PMWS)以及其它相關臨床症狀之主要病原，PCV2所引起的各種臨床症狀被統稱為第二型猪環狀病毒症（PCV2 associated diseases, PCVAD）。PCV2普遍感染於台灣的豬場，但此病毒演化相關資料相當少見；而新穎的分子診斷方法迴路媒介恆溫增幅法（loop-mediated isothermal amplification, LAMP）亦較少使用於此病毒的診斷，包括普遍汙染於PK細胞株的第一型豬環狀病毒(porcine circovirus type 1, PCV1)。因此，本篇論文的目的是進行台灣PCV2病毒的分子流行病學研究，並建立新的臨床診斷技術：迴路媒介恆溫增幅法用以診斷豬環狀病毒（PCV）。在第一項研究中，將2001年至2011年間收集的571株台灣PCV2分離株之基因體全長以聚合酶鏈反應(polymerase chain reaction, PCR)加以增幅並將其產物直接定序。以演化樹分析定序之結果，可發現這些分離株主要分為兩種不同的基因型，PCV2a和PCV2b。其中131株分離株之基因型屬於PCV2a，並可被進一步區分為2B、2D以及2E等3個基因亞型；另外440分離株之基因型屬於PCV2b，並可被進一步區分為1A、1B以及1C等3個基因亞型。 在2001年台灣PCV2分離株主要之基因型為PCV2a，然而自2003年開始，台灣PCV2分離株主要基因型為PCV2b。經由該研究結果分析，台灣PCV2分離株之主要基因型已從PCV2a轉變為PCV2b。在第二項研究中，主要以PCV2第二開放讀碼區(open reading frame 2, ORF2)基因序列為基礎，開發出結合即時監控系統(real-time monitoring system)之LAMP檢測法用以檢測臨床檢體中是否有PCV2a或PCV2b等不同基因型PCV2病毒之單獨或混和感染。LAMP檢測法可以在50分鐘內完成反應，並可同時檢測臨床樣品中PCV2a和PCV2b等不同基因型之分離株，而且並無交叉反應。以LAMP及巢式聚合酶鏈反應（nested PCR）共同檢測含PCV2a和PCV2b基因序列的標準重組質體（recombinant plasmid），LAMP和nested PCR之最低檢出濃度皆為10 copies/μl，顯示LAMP之敏感度等同於nested PCR。將168例臨床檢體同時以LAMP與nested PCR進行檢測，並將nested PCR檢測結果作為金標準（gold standard）以進行比較，LAMP之相對靈敏度（relative sensitivity）為97.7%，相對特異性（relative specificity）為100%。綜合上述研究結果顯示LAMP檢測法可作為一種簡便、快速鑑別PCV2a與PCV2b之診斷工具。在第三項研究中，我們開發出結合real-time monitoring system之LAMP檢測法以檢測PCV1，並應用此法檢測PCV1是否汙染豬用疫苗。LAMP反應可以在一小時內完成，且對於PCV1具有高度特異性，對於其它常見豬隻病毒並不會產生交叉反應。以LAMP及nested PCR共同檢測含PCV1基因序列的recombinant plasmid，LAMP和nested PCR之最低檢出濃度皆為10 copies/μl，顯示LAMP之敏感度等同於nested PCR。另一方面我們收集 25個豬用疫苗並以LAMP和nested-PCR同時進行檢測，其中3個豬用疫苗（11%, 3/25）可同時被LAMP和nested-PCR檢出含PCV1的DNA。LAMP檢測法可作為一個高度特異，敏感，快速診斷技術，並可有效檢測出豬用疫苗是否遭到PCV1 DNA之汙染，為生物製劑之品質做好把關之工作。

關鍵字

豬環狀病毒 ； 迴路媒介恆溫增幅法 ； 樹狀圖分析

並列摘要

Porcine circovirus type 2 (PCV2) is one of the major swine viral diseases and caused significant economic loss to pig producers worldwide, including Taiwan. PCV2 has been considered as the causative agent of postweaning multisystemic wasting syndrome (PMWS) as well as other clinical diseases. All these associated syndromes have been categorized as PCV2 associated diseases (PCVAD). PCV2 infection is commonly present in pig farms in Taiwan; however, the information regarding evolution and phylogenic analysis of PCV2 in this area is rare. In addition, application of loop-mediated isothermal amplification (LAMP) in PCV infection, including commonly PK cell line-contaminated PCV1, has rarely been reported. The purposes of this thesis were to perform molecular epidemiological studies of PCV2 in Taiwan and establish a novel diagnostic tool, LAMP, to detect PCV. In the first study, complete genomes of PCV2 were amplified by polymerase chain reaction (PCR) and sequenced directly from 571 Taiwanese PCV2 isolates collected during the period of 2001 and 2011. The phylogenetic tree analysis indicated that these isolates could be divided into 2 distinct genotypes, PCV2a and PCV2b. Among the 571 isolates, 131 isolates were clustered within genotype PCV2a (further classified as 2B, 2D, and 2E), and 440 isolates were clustered within genotype PCV2b (further classified as 1A, 1B, and 1C). PCV2a genotype predominated in 2001, and then PCV2b became the major prevalent genotype since 2003. In the second study, a LAMP method with a real-time monitoring system was developed based on open reading frame 2 (ORF2) in the viral genome for the simultaneous detection and differentiation of PCV2a and PCV2b in clinical samples. The LAMP reaction could be completed within 50 min, and the PCV2a and PCV2b could be detected and accurately distinguished without cross-reaction. The detection limit of the LAMP were 10 copies/μl of the PCV2a and PCV2b recombinant plasmids, demonstrating detection limit comparable to that obtained using nested polymerase chain reaction (nested PCR). On the basis of the results of 168 clinical samples using nested PCR as the gold standard, the relative sensitivity of LAMP was 97.7% and the relative specificity was 100%. Thus, LAMP can be a simple, rapid and valuable tool for the differential diagnosis of PCV2a and PCV2b. In the third study, we have developed a LAMP method with a real-time monitoring system for the detection of PCV type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1, no cross-reaction to other common swine viral pathogens was observed and the LAMP reaction could be completed within one hour. The detection limit of the LAMP for PCV1 DNA was 10 copies/μl of the positive recombinant plasmid, comparable to that obtained from nested PCR. In addition, 25 commercial swine vaccines were tested by both LAMP and nested PCR, and PCV1 DNA was detected in 3/25 commercial swine vaccines (11%) by either method. The LAMP method holds promise for using as a highly specific, sensitive, and rapid diagnostic assay for PCV1 DNA detection in commercial swine vaccines.

並列關鍵字

Porcine circovirus ； loop mediated isothermal amplification ； phylogenetic tree analysis

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