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研究生:郭依婧
研究生(外文):Yi-Ching Kuo
論文名稱:豬鐵士古病毒地方性感染之血清型分子鑑定
論文名稱(外文):Molecular Serotyping of the Porcine Teschoviruses in Endemically Infected Pigs
指導教授:王汎熒
指導教授(外文):Fun-In Wang
口試委員:張本恆張家宜李璠
口試日期:2015-07-15
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子暨比較病理生物學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:50
中文關鍵詞:豬鐵士古病毒血清型反轉錄聚合酶連鎖反應
外文關鍵詞:teschovirusserotypingRT-PCR
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豬鐵士古病毒(Porcine Teschovirus, PTV)為Picornaviridae科Teschovirus屬,直徑約為25-30nm、球型、正向單股RNA病毒。目前PTV有13種血清型,各年齡豬隻皆有感受性,臨床症狀以引起神經症狀之腦脊髓灰質炎及下痢為主。台灣在2000年及2004年爆發兩次疫情,現今在豬場呈現普遍的地方性感染。先前檢測出台灣存有五種血清型,分別為PTV-1、-4、-6、-7、-11,最常見的為PTV-7及PTV-6。本研究目的為利用增幅病毒VP1核酸序列進行鑑定台灣目前PTV是否有更多血清型的存在。實驗樣材來自於七個豬場,每場採集3隻淘汰病弱豬及1隻臨床症狀健康豬隻;每頭豬隻收集糞便、迴腸、扁桃、鼠蹊淋巴結、血漿、脾臟、腎臟、膀胱、尿液以及鼻腔拭鏡共10個樣材。首先以增幅5’NTR高度保留區域檢測PTV感染,所有28頭豬隻皆為PTV陽性(100%, 28/28),其中又以糞便的陽性率最高(96%, 27/28),其次為迴腸、扁桃、鼻腔試鏡(93%, 26/28),血漿(86%, 24/28),鼠蹊淋巴結及尿液(79%, 22/28),膀胱(64%, 18/28),脾臟(57%, 16/28)及腎臟(10%, 10/28)。接著利用本研究新設計之引子增幅病毒VP1序列,在217個PTV陽性樣本中有63個成功完成增幅且區分血清型。28頭豬隻皆至少有一種血清型存在,總共檢測出五種PTV血清型,分別為PTV-2、-4、-5、-6、-10,其中又以PTV-10 (57%, 36/63)最為常見,其次為PTV-2 (14/63, 22%)。其中有14頭豬隻有兩種以上血清型存於不同臟器 (14/28, 50%)。本研究再度印證了台灣PTV普遍感染的現象,加上先前的結果,證實台灣目前至少存在8種血清型,分別為PTV-1、-2、-4、-5、-6、-7、-10、-11。值得注意的是在分別來自三個豬場的三頭豬隻尿液亦可檢測出PTV-10,顯示PTV可經尿液排出體外,糞尿混合下加速了病毒傳播速度,證實之前的推論。

Porcine teschoviruses (PTVs) is a non-enveloped, spherical, positive-sense, single-stranded RNA virus, which belong to genus Teschovirus within the family Picornaviridae. Up to date, PTVs are reclassified into 13 serotypes that are associated with a variety of clinical signs and prominent with polioencephalomyelitis and diarrhea. Two epidemic outbreaks of PTV-1 occurred in Taiwan, one in 2000 and a second in 2004, causing severe economic loss. The enzootic status is now confirmed in swine herds in Taiwan. Previous study has demonstrated at least 5 different serotypes of PTV (PTV-1, -4, -6, -7, -11) existing in Taiwan, and PTV-7 and -6 are the most common serotypes. The aim of this study was to investigate the wider variety of serotypes of PTV by introducing a set of newly designed primer, based on the sequence of VP1 outer capsid gene presented in the feces, plasma, urine, nasal swab, and solid samples in naturally infected piglets. Samples were collected from 7 clinically healthy and 21 culled piglets of 7 different herds. The PTV infections were screened by the published primers to amplify highly conserved 5’NTR region using RT-PCR followed by nested PCR. The PTV detection rate was 100% (28/28) by heads. Out of 217/280 PTV-positive samples the most common detected sample was feces (96%, 27/28), and followed by in decreasing order ileum, tonsil, and nasal swab (93%, 26/28), plasma (86%, 24/28), inguinal LN and urine (both were 79%, 22/28), bladder (64%, 18/28), spleen (57%, 16/28), and kidney (10%, 10/28). VP1 region was successfully amplified, by the newly designed primers, out of 63/217 (29%) pan-PTV nested PCR-positive samples, sequenced, and phylogenetic tree constructed. All 28 piglets had at least one serotype existed, and a total of 5 serotypes of PTV-2, PTV-4, PTV-5, PTV-6, and PTV-10, were identified from 28 piglets. The most common serotype was PTV-10 (36/63, 57%) and the 2nd one was PTV-2 (14/63, 22%). In 14/28 (50%) heads had at least two serotypes identified in the same animals but in different samples. In 2/7 herds, four PTV serotype co-circulated. In conclusion, this study confirms the enzootic and contaminated status of swine herds, combined with previous study at least 8 serotypes of PTV, namely PTV-1、-2、-4、-5、-6、-7、-10、-11 are identified. In particular PTV-10 is found shed in the urine of 3 piglets from 3 separate herds, consistent with the multiple models of PTV pathogenesis. Urine makes solid feces into semisolid slurry thus enhancing the exposure of piglets to PTV.

口試委員審定書 i
致謝 ii
中文摘要 iii
ABSTRACT iv
CONTENTS v
LIST OF FIGURES ix
LIST OF TABLES x
LIST OF APPENDICES xi
Chapter 1 Introduction 1
Chapter 2 Literature Review 3
2.1 Porcine teschovirus 3
2.1.1 Taxonomy and classification 3
2.1.2 Morphology, genome and capsid porteins 3
2.2 Epidemiology 4
2.2.1 Epidemiology in Taiwan 4
2.3 Clinical signs 5
2.3.1 Polioencephalomyelitis 5
2.3.2 Reproductive diseases 6
2.3.3 Diarrhea 6
2.3.4 Pneumonia, pericarditis, and myocarditis 6
2.4 Pathological changes 6
2.5 Pathogenesis 7
2.6 Detection of PTVs by Reverse transcription polymerase chain reaction (RT-PCR) and Nested PCR 8
2.7 Serotyping and Molecular serotyping (Genotyping) of PTVs 8
2.7.1 Serotyping of PTVs 8
2.7.2 Molecular serotyping (Genotyping) of PTVs 8
2.7.3 Whole P1 region 9
2.7.4 VP1 region 10
2.7.5 VP2 region 10
Chapter 3 Materials and Methods 11
3.1 Sample preparations 11
3.1.1 Animals 11
3.1.2 Sampling for RT-PCR 11
3.2 Sources and cell culture of prototype virus 11
3.3 RNA extraction 12
3.3.1 RNA extraction from solid tissues 12
3.3.2 RNA extraction from fluid samples (diluted feces, urine, plasma, and nasal swab) 13
3.4 Primer selection 14
3.4.1 Porcine teschoviruses 14
3.4.2 Primers for PTVs molecular serotyping on VP1 14
3.5 Reverse transcription polymerase chain reaction (RT-PCR) for 5''NTR (pan-PTV) and VP1 18
3.6 Nested PCR for PTV 5''NTR and VP1 18
3.7 Gel electrophresis 19
3.7.1 Gene sequencing and data analysis for VP1 19
Chapter 4 Results 21
4.1 Sample collection 21
4.2 Screening for PTVs infection in endemic infected pigs 21
4.2.1 PTVs detection by RT-PCR followed by nested PCR 21
4.3 Molecular serotyping of PTVs based on VP1 sequence 22
4.3.1 VP1 amplification of reference strains 22
4.3.2 Genotyping of VP1 region in samples 22
Chapter 5 Discussion 24
REFERENCES 30
FIGURES 35
TABLES 39
APPENDICES 44


呂榮修, 蔡義雄, 鍾明華, 劉培柏, 李永林, 楊喜吟, 王金和, 王吉德, 1981. 台灣豬腸道病毒分離與抗體調查. 台灣省畜衛試研報 17, 83-90.
Alexandersen, S., Knowles, N.J., Dekker, A., Belsham, G.J., Zhang, Z., Koenen, F. 2012. Piconavirus, In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.) Diseases of Swine. Wiley-Blackwell, pp. 587-620.
Anonymous. 2008. Teschovirus Encephalomyelitis (Previously Enterovirus Encephalomyelitis or Teschen/Talfan Disease), In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. World Organisation for Animal Health, Paris, pp. 1146-1152.
Bangari, D.S., Pogranichniy, R.M., Gillespie, T., Stevenson, G.W., 2010. Genotyping of porcine teschovirus from nervous tissue of pigs with and without polioencephalomyelitis in Indiana. J. Vet. Diagn. Invest. 22, 594-597.
Boros, A., Nemes, C., Pankovics, P., Kapusinszky, B., Delwart, E., Reuter, G., 2012. Porcine teschovirus in wild boars in Hungary. Arch. Virol. 157, 1573-1578.
Buitrago, D., Cano-Gomex, C., Aguero, M., Fernandez-Pacheco, P., Gomez-Tejedor, C., Jimenez-Clavero, M.A., 2010. A survey of porcine picornaviruses and adenoviruses in fecal samples in Spain. J. Vet. Diag. Invest. 22, 763-766.
Cano-Gomez, C., Garcia-Casado, M.A., Soriguer, R., Palero, F., Jimenez-Clavero, M.A., 2013. Teschoviruses and sapeloviruses in faecal samples from wild boar in Spain. Vet. Microbiol. 165, 115-122.
Cano-Gomez, C., Palero, F., Buitrago, M.D., Garcia-Casado, M.A., Fernandez-Pinero, J., Fernandez-Pacheco, P., Aguero, M., Gomez-Tejedor, C., Jimenez-Clavero, M.A., 2011b. Analyzing the genetic diversity of teschoviruses in Spanish pig populations using complete VP1 sequences. Infect. Genet. Evol. 11, 2144-2150.
Chiu, S.C., Hu, S.C., Chang, C.C., Chang, C.Y., Huang, C.C., Pang, V.F., Wang, F.I., 2012. The role of porcine teschovirus in causing diseases in endemically infected pigs. Vet. Microbiol. 161, 88-95.
Chiu, S.C., Yang, C.L., Chen, Y.M., Hu, S.C., Chiu, K.C., Lin, Y.C., Chang, C.Y., Wang, F.I., 2014. Multiple models of porcine teschovirus pathogenesis in endemically infected pigs. Vet. Microbiol. 168, 69-77.
Deng, M.Y., Millien, M., Jacques-Simon, R., Flanagan, J.K., Bracht, A.J., Carrillo, C., Barrette, R.W., Fabian, A., Mohamed, F., Moran, K., Rowland, J., Swenson, S.L., Jenkins-Moore, M., Koster, L., Thomsen, B.V., Mayr, G., Pyburn, D., Morales, P., Shaw, J., Burrage, T., White, W., McIntosh, M.T., Metwally, S., 2012. Diagnosis of porcine teschovirus encephalomyelitis in the Republic of Haiti. J. Vet. Diagn. Invest. 24, 671-678.
Donin, D.G., de Arruda Leme, R., Alfieri, A.F., Alberton, G.C., Alfieri, A.A., 2014. First report of Porcine teschovirus (PTV), Porcine sapelovirus (PSV) and Enterovirus G (EV-G) in pig herds of Brazil. Tropical Anim. Hlth. Produc. 46, 523-528.
Harding, J.D.J., Done, J.T., Kershaw, G.F., 1957. A transmissible polioencephalomyelitis of pigs (Talfan disease). Vet. Rec. 69, 824-832.
Holman, J.E., Koestner, A., Kasza, L., 1966. Histopathogenesis of porcine polioencephalomyelitis in the germ free pig. Pathol. Vet. 3, 633-651.
Horstmann, D.M., 1952. Experiments with Teschen disease (virus encephalomyelitis of swine). J. Immunol. 69, 379-394.
Huang, T.S., Wang, C., Deng, M.C., Jeng, J.J., Lee, S.H., Pan, C.S., Lee, M.C., Jong, M.H., 2009. The results of virus isolation in swine tissue samples submitted by LDCC and tested by viral isolation and PCR and/or RT-PCR in 2008. Exp Rep. AHRI (Animal Health Reserch Institute) 44, 35-46.
Jimenez-Clavero, M.A., Fernandez, C., Ortiz, J.A., Pro, J., Carbonell, G., Tarazona, J.V., Roblas, N., Ley, V., 2003. Teschoviruses as indicators of porcine fecal contamination of surface water. Appl. Environ. Microbiol. 69, 6311-6315.
Kaku, Y., Murakami, Y., Sarai, A., Wang, Y., Ohashi, S., Sakamoto, K., 2007. Antigenic properties of porcine teschovirus 1 (PTV-1) Talfan strain and molecular strategy for serotyping of PTVs. Arch. Virol. 152, 929-940.
Kaku, Y., Sarai, A., Murakani, Y., 2001. Genetic reclassification of porcine enteroviruses. J. Gen. Virol. 82, 417-424.
Knowles, N.J., Buckley, L.S., Pereira, H.G., 1979. Classification of porcine enteroviruses by antigenic analysis and cytopathic effects in tissue culture: Description of 3 new serotypes. Arch.Virol. 62, 201-208.
Kuo, CC., 2014. Urinary shedding of porcine teschovirus in endemically infected field situation. National Taiwan University Master degree thesis.
La Rosa, G., Muscillo, M., Di Grazia, A., Fontana, S., Iaconelli, M., Tollis, M., 2006. Validation of rt-PCR assays for molecular characterization of porcine teschovirus and enteroviruses. J. Vet. Med. B Infect. Dis. Vet. Public Health 53, 257-265.
Qiu, Z., Wang, Z., Zhang, B., Zhang, J., Cui, S., 2013. The prevalence of porcine teschovirus in the pig population in northeast of China. J. Virol. Methods 193, 209-214.
Racaniello, V.R., 2006. One hundred years of poliovirus pathogenesis. Virology 344, 9-16.
Radstrom, P., Knutsson, R., Wolffs, P., Dahlenborg, M., Lofstrom, C. 2003. Pre-PCR processing of samples. In Sachse, K. and Frey, J. (Eds.) PCR detection of microbial pathogens, pp. 31-50.
Sachse, K. 2003. Specificity and performance of diagnostic PCR assays. In Sachse, K. and Frey, J. (Eds.) PCR detection of microbial pathogens, pp. 3-29.
Salles, M.W., Scholes, S.F., Dauber, M., Strebelow, G., Wojnarowicz, C., Hassard, L., Acton, A.C., Bollinger, T.K., 2011. Porcine teschovirus polioencephalomyelitis in western Canada. J. Vet. Diagn. Invest. 23, 367-373.
Smith, T.J., Baker, T., 1999. Picornaviruses: epitopes, canyons, and pockets. Adv. Virus Rec. 52, 1-23.
Sozzi. E., Barbieri, I., Lavazza, A., Lelli, D., Moreno, A., Canelli, E., Bugnetti, M., Cordioli, P., 2010. Molecular characterization and phylogenetic analysis of VP1 of porcine enteric picornaviruses isolates in Italy. Transbound. Emerg. Dis. 57, 434-442.
Wang, B., Tian, Z.J., Gong, D.Q., Li, D.Y., Wang, Y., Chen, J.Z., An, T.Q., Peng, J.M., Tong, G.Z., 2010. Isolation of serotype 2 porcine teschovirus in China: evidence of natural recombination. Vet. Microbiol. 146, 138-143.
Yamada, M., Kozakura, R., Nakamura, K., Yamamoto, Y., Yoshii, M., Kaku, Y., Miyazaki, A., Tsunemitsu, H., Narita, M., 2009. Pathological changes in pigs experimentally infected with porcine teschovirus. J. Comp. Pathol. 141, 223-228.
Zell, R., Dauber, M., Krumbholz, A., Henke, A., Birch-Hirschfeld, E., Stelzner, A., Prager, D., Wurm, R., 2001. Porcine teschoviruses comprise at least eleven distinct serotypes: molecular and evolutionary aspects. J.Virol. 75, 1620-1631.
Zell, R., Krumbholz, A., Henke, A., Birch-Hirschfeld, E., Stelzner, A., Doherty, M., Hoey, E., Dauber, M., Prager, D., Wurm, R., 2000. Detection of porcine enteroviruses by nRT-PCR: differentiation of CPE groups I-III with specific primer sets. J. Virol. Methods 88, 205-218.


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