以原位雜交偵測豬淋巴組織內豬纖鍊病毒及其致病機轉之探討

李遙

doi:10.6342/NTU.2012.01394

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以原位雜交偵測豬淋巴組織內豬纖鍊病毒及其致病機轉之探討

Detection and Pathogenesis of Torque Teno Sus Virus in Swine Lymphoid Tissues by in Situ Hybridization

李遙 , Masters　　Advisor：龐飛　　Co-advisor ：鄭謙仁

繁體中文 DOI： 10.6342/NTU.2012.01394

豬纖鍊病毒；豬環狀病毒第二型；豬離乳後多系統消耗症候群；豬環狀病毒第二型相關疾病；原位雜交；組織微陣列； TTSuV ； PCV2 ； PMWS ； PCVAD ； ISH ； TMA

Abstract │ Reference (101) │ Altmetrics

Abstract 〈TOP〉

豬纖鍊病毒(torque teno sus virus, TTSuV)屬於Anelloviridae科，為無封套DNA病毒，具有負向單股環狀DNA基因體，序列具高度變異性。目前有兩種TTSuV，分別是豬纖鍊病毒第一型(torque teno sus virus 1, TTSuV1)與第二型(torque teno sus virus 2, TTSuV2)，感染範圍遍及全球養豬國家；TTSuV1於台灣豬場豬隻陽性率為96.88%(465/480)(徐，2010)，而TTSuV2則為56.25%(270/480)(黃，2010)。TTSuV如同豬小病毒(parvovirus, PPV)、豬生殖與呼吸症候群病毒(porcine reproductive and respiratory syndrome virus, PRRSV)，被認為與豬環狀病毒第二型(porcine circovirus type 2, PCV2)共同感染而導致包括豬離乳後多系統消耗症候群(postweaning multisystemic wasting syndrome, PMWS)在內的豬環狀病毒相關疾病(porcine circovirus-associated disease, PCVAD)，然而關於TTSuV的細胞親合性與致病機制的相關資料則甚少。在本研究中，首先建立於組織中偵測TTSuV之原位雜交(in situ hybridization, ISH)技術，並以田間耗弱豬隻鼠蹊淋巴結為樣本，進行重組製成組織微陣列(tissue microarray, TMA)，結合免疫化學(immunohistochmistry, IHC)染色，進行多重病毒性病原及各類免疫細胞之偵測，以辨別TTSuV之標的細胞；接著，應用自動化陽性圖素判讀結合統計分析，來調查TTSuV與其他病毒性因子如PCV2、PPV、與PRRSV間的關係，探討TTSuV是否與PMWS或PCVAD有關。鏡檢下發現TTSuV病毒核酸多出現於淋巴小節結之外圍帶與淋巴小節結間區，高倍觀察陽性細胞為淋巴球與漿細胞；鏡檢結果與鄧肯分組分析(Duncan’s multiple range test)皆顯示，淋巴球增生出現於TTSuV1、2輕微感染階段，淋巴球流失則在PCV2中等以上感染中出現，肉芽腫性炎症反應出現於TTSuV1、2以及PCV2中等至嚴重程度感染的樣本中。複迴歸分析(multiple regression analysis)進一步指出，TTSuV1對於B淋巴球增生有顯著效應(P< 0.05)，TTSuV2對於巨噬細胞浸潤有顯著效應(P< 0.05)，而PCV2與B淋巴球流失、T淋巴球以及巨噬細胞增生有顯著效應(P< 0.05)。相關性統計分析(Pearson correlation analysis)顯示，TTSuV2與PCV2的病毒量有顯著正相關(r= 0.236, P< 0.05)，不過卡方分析(chi square test)顯示TTSuV1、2與PCV2的感染無顯著關聯(P> 0.05)，暗示TTSuV感染雖然與PCV2無關，但在共同感染的情形下雙方有協同增殖的關係。根據本研究結果，推測TTSuV在受刺激活化的淋巴球內增殖，隨後PCV2與其他病原逐漸入侵感染並共同影響免疫細胞；而相對於普遍分布的TTSuV1，TTSuV2被懷疑較具致病性，可能與PCV2共同引發類似PMWS的組織病變–肉芽腫性炎症反應。本實驗之結果證實TTSuV1與TTSuV2是一種常見的PCV2共同感染因子，且伴隨PCV2的感染而導致PMWS或是PCVAD的產生，而這可能肇因於PCV2與TTSuV交互對不同種免疫細胞所造成之變化。

Parallel Abstract 〈TOP〉

Torque teno sus virus (TTSuV), belonging to Anelloviridae, is a non-enveloped DNA virus that has a circular, single-stranded, negative sense genome with high sequence diversity. TTSuV contains two major groups, Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2), infecting pigs world-wide. The prevalence of TTSuV1 in Taiwan was 96.88% (465/480) (Shyu, 2010), while the prevalence of TTSuV2 in Taiwan was 56.25% (270/480) (Huang, 2010). As porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV), TTSuV has been suggested as a co-factor with porcine circovirus type 2 (PCV2) infection belonging to porcine circovirus-associated diseases (PCVAD), including postweaning multisystemic wasting syndrome (PMWS). However, information regarding the cell tropism and pathogenesis of TTSuV is rather limited. In the present study, in situ hybridization (ISH) combined with tissue microarray (TMA) for the detection of TTSuVs in swine inguinal lymph nodes was established in order to identify the target cells. Subsequently, automatic positive pixel evaluation combined with statistical analysis was applied to investigate the association between TTSuV and other porcine viral factors including PCV2, PPV, and PRRSV which were related with PMWS or PCVAD. The histological results revealed that viral nucleic acid of TTSuV was mainly located in the mantle zone and interfollicular region of lymph nodes. In high magnification, the TTSuV-positive cells in ISH were lymphocytes and plasma cells. According to microscopic observation accompanied with Duncan’s multiple range test, lymphocyte proliferation was present in mild infections of TTSuV1 and TTSuV2, whereas lymphocyte depletion was present in moderate infection of PCV2. Granulomatous inflammation was noted among moderate to severe infections of TTSuV1, TTSuV2, and PCV2. Furthermore, the results of multiple regression analysis demonstrated that TTSuV1 had significant effect for B lymphocyte proliferation (P< 0.05), while TTSuV2 had significant effect for macrophage infiltration (P< 0.05). PCV2, on the other hand, had multiple significant effects for B lymphocyte depletion, T lymphocyte proliferation, and macrophage proliferation (P< 0.05). In addition, Pearson correlation analysis indicated that there was significantly positive correlation between the viral loads of TTSuV2 and PCV2 (r= 0.236, P< 0.05). Nevertheless, chi square tests showed that there was no relation between infections of TTSuV and PCV2 (P> 0.05). The results implied that the collaboration between the viral loads of TTSuV and PCV2 was discernible when in co-infection, though there was no significant relationship among TTSuV and PCV2 infection rates. Based on the present study, TTSuV was suspected to replicate in stimulated and activated lymphocytes, followed by infections of PCV2 and other pathogens that brought about further fluctuation of various immunocytes. Compared with ubiquitous distribution of TTSuV1, TTSuV2 was assumed to be more pathogenetic, and it might co-infect with PCV2 to cause granulomatous inflammation, which is suggested as a PMWS-like lesion. The results may support that TTSuV1 and TTSuV2 are common co-factors with PCV2 in PMWS or PCVAD, and that could contribute to some fluctuations in various populations of immune cells.

Reference ( 101 ) 〈TOP〉

李育宗。豬增生性壞死性肺炎病灶與常見病毒包括第二型豬環狀病毒、豬生殖與呼吸道綜合症病毒、豬流行性感冒病毒及豬小病毒之關聯性。國立台灣大學獸醫學研究所碩士論文，2005。
連結：
蕭君倪。建立偵測常見豬隻呼吸道病毒性豬隻病毒性疾病即時聚合酶鍊鎖反應技術。國立台灣大學獸醫學研究所碩士論文，2010。
連結：
張志成，黃安進，徐寶如，馬尉真，龐飛，王建雄。2008至2009年台灣豬場Torque Teno Virus (TTV)流行概況之探討。台灣獸醫誌Taiwan Vet J 37: 45-52，2011。
連結：
Allan, G.M., Ellis, J.A., 2000. Porcine circoviruses: a review. J Vet Diagn Invest 12, 3-14.
連結：
Aramouni, M., Segales, J., Cortey, M., Kekarainen, T., 2010. Age-related tissue distribution of swine Torque teno sus virus 1 and 2. Vet Microbiol 146, 350-353.
連結：

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