定量地方感染豬隻組織內之豬鐵士古病毒含量

Quantification of Porcine Teschovirus Load in Tissues of Endemically Infected Pigs

楊智麟(Chih-Lin Yang)

指導教授 ： 王汎熒

國立臺灣大學/獸醫專業學院/分子暨比較病理生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01615

摘要

中文摘要
豬鐵士古病毒 (Porcine teschovirus, PTV) 在病毒分類學上屬於 (PTV) Picornaviridae科Teschovirus屬。為一直徑約25~30 nm球形、無封套、正向單股的RNA病毒。PTV目前普遍存在於臺灣豬場，且呈現地方感染。感染豬隻大部分以亞急性至慢性感染並持續排毒，體內含有病毒但未發病，病毒與免疫反應共存，故各臟腑的病毒含量應維持一接近穩定的狀態。之前的研究將豬之組織分為四大類，包含腦，內臟器官，淋巴器官及消化道，使用nested RT-PCR偵測，顯示PTV在消化道有較高偵測機率，而於淋巴器官至腦部則遞減。故推測PTV可能致病機轉為病毒先感染腸道並進行増殖，隨後穿透至局部淋巴結，再進一步形成病毒血症導致各臟腑感染，最後再穿透血腦障壁抵達中樞神經，此結果與人類小兒痲痺病毒的fecal-oral model致病模式基本上吻合但也有差異之處。本研究目的為運用即時聚合酶鏈鎖反應 (qPCR) 定量PTV在豬隻組織內之病毒含量，方法為以絕對定量 (Absolute quantification) 偵測選定之七種部位 (腦部、腎臟、脾臟、扁桃腺、迴腸淋巴結、迴腸、鼠蹊淋巴結) 的病毒含量。並期望能與建立之致病機轉比較。在製作標準曲線方面選用兩種血清型病毒株，分別為淡水家畜衛生試驗所於西元2004所分離之PTV-1 (臺灣PTV的prototype)；另外則選用基因序列上與PTV-1 prototype (Talfan strain) 有高度相似性之PTV-7 reference strain WR-2以比較兩者之敏感性。結果顯示PTV-1與PTV-7最低濃度皆可偵測至101 copies/μg RNA。PTV-1 至11皆可被増幅；但Sapelovirus、PEV-9及PEV-10則呈現陰性，顯示引子對PTV具有良好專一性。針對樣品RNA之偵測，七種臟器中以鼠蹊淋巴結具有最高的病毒含量，若以PTV-1為標準測得約含有103.07±1.81 copy/μg RNA；若以PTV-7為標準測得為102.81±1.81 copy/μg RNA迴腸與迴腸淋巴結次之，腦部病毒量最少(PTV-1: 102.43±0.390;PTV-7: 102.17±0.4)。此結果與所建構的致病機轉基本一致，迴腸含量較預期少，推測可能的原因與腸道上皮的半衰期較短以及病毒隱匿於淋巴結利於病毒血症的發展維持有關。而腦部含量較預期多，推測可能與病毒由鼻腔及扁桃線上行感染有關。
關鍵字：鐵士古病毒、地方感染豬隻、即時聚合酶鏈鎖反應

關鍵字

鐵士古病毒 ； 地方感染豬隻 ； 即時聚合酶鏈鎖反應

並列摘要

Abstract
Porcine teschoviruses (PTVs) belong to genus Teschovirus within the family Picornaviridae. The virions are characterized by 25 to 30 nm in diameter, spherical, nonenveloped with a linear plus sense ssRNA genome. In Taiwan, PTVs are common seen in pig herds in endemic status. The most pigs show subacute to chronic infection and persistent shedding virus without clinical signs though viruses are present within the body. Because virus and immune response coexists, thus viral load within organs and tissue might maintain a nearly stable state. Previous studies divided the organs and tissues of endemically infected into four major groups, including brains, visceras, lymphoid organs, and digestive tracts, and detected virus through nested RT-PCR. The results revealed that the PTVs within digestive tracts had higher detection rates, and the rates decreased from lymphoid organs to brain tissues. Thus a possible pathogenesis would be that PTV firstly infects intestine and propagate, then penetrates to regional lymph nodes followed by viremia with subsequent infection of visceral organs and tissues, and the virus finally penetrates through the blood brain barrier (BBB) and reaches the central nervous system (CNS). These results generally coincide with the fecal-oral model pathogenesis of human poliovirus although there are still some differences between human poliovirus and Porcine PTV infections. The aim of study was to carry out real-time polymerase chain reaction (qPCR) to absolute quantitate viral load within porcine tissues, including brain, kidney, spleen, tonsil, ileac lymph node (LN), ileum, and inguinal LN. Because PTVs have 11 serotypes, two virus PTV-1 and PTV-7 Two virus strains were chosen to construct standard curve and to compare sensitivity. The PTV-1 was isolated by Animal health research institute in 2004 (Prototype of PTV in Taiwan). The PTV-7 WR-2 strain was chosen due to high similarity of genetic sequence with Talfan strain (PTV-1 prototype) and previous outbreaks in Taiwan were similar to or typical of Talfan disease. The qRT-PCR revealed the detection limit of PTV-1 and PTV-7 standard was 101 copies/μg RNA. The qRT-PCR could successfully amplify PTV-1 to 11 whereas Sapelovirus, PEV-9 and PEV-10 were negative, indicating the primers have good specificity for PTVs. Inguinal LN had highest viral load among the seven organs, containing 103.07±1.81 copy/μg RNA (PTV-1 standard) or 102.81±1.81 copy/μg RNA (PTV-7 standard). Ileum and ileac LN had fewer viral load, and the brain had the least viral load (102.43±0.390: PTV-1; 102.17±0.4: PTV-7). In general, the results were consistent with the fecal-oral model of pathogenesis. The lower viral load in ileum than inguinal LN may be due to the shorter half-life of intestinal epithelium while the virus harbored within the LN may be favorable for the viral sustainability within the animal body. On the contrary, viral load in brain was higher than expected consistent with the routes of intranasal infection and ascending infection from the tonsil, in addition to the well-known fecal-oral route.
Key words: Teschovirus, PTV, endemically infected pigs, qRT-PCR, viral load

並列關鍵字

Teschovirus ； PTV ； endemically infected pigs ； qRT-PCR ； viral load

參考文獻

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