臺灣博碩士論文加值系統

家禽流行性感冒和鴨肝炎是家禽重要的病毒性疾病。這兩種疾病傳播快速，可造成感染的家禽大量死亡，使家禽產業蒙受巨大的經濟損失。家禽流行性感冒病毒和鴨肝炎病毒皆為單股RNA病毒，容易因環境壓力發生變異。本論文將對近年來台灣發生的家禽流行性感冒和鴨肝炎病毒基因進行研究。第一部分:分析台灣2013至2015年，感染雞隻的H5N2病毒的血球凝集蛋白切割位胺基酸序列與致病力的關係。2003年台灣雞群首次爆發H5N2病毒的感染，至2013年已發現三種血球凝集蛋白切割位胺基酸序列: QREKR/GL、QRKKR/GL和QRRKR/GL。於2013和2014年分離的病毒，再發現兩種新的血球凝集蛋白切割位胺基酸序列，分別是QKEKR/GL和KREKREKR/GL，對雞的致病性分別屬於低致病性和高致病性；2015年發現的病毒其血球凝集蛋白切割位胺基酸序列皆為QREKR/GL，皆為低致病性。為了瞭解台灣低致病性H5N2病毒演化為高致病性病毒的機制，將2003年低致病性H5N2病毒接種於雞胚胎蛋中連續繼代，於第10代的尿囊液，可發現四種不同的血球凝集蛋白切割位胺基酸序列。台灣的H5N2病毒可藉由點突變、插入和刪除基因的方式改變血球凝集蛋白切割位胺基酸序列和致病力。這些發現有助於更了解台灣雞群中的H5N2病毒血球凝集蛋白切割位胺基酸序列的變化機制並作為防疫的參考。第二部分: 2010至2014年感染台灣鴨場的鴨肝炎病毒序列的研究。從52個小鴨臨床病例中，分離到10株鴨肝炎病毒，陽性率為19% (10/52)。分析病毒5’-UTR基因序列，顯示病毒皆屬於第一型鴨A型肝炎病毒。根據病毒VP1胺基酸序列的親緣分析結果，發現病毒可被分成兩個基因群，分別稱為台灣基因一型和台灣基因二型。台灣基因一型的病毒(8株)與2003年台灣發現的病毒株03D同屬一群；台灣基因二型的病毒(2株)則相似於中國發現的病毒株。兩個基因群彼此之間胺基酸序列的差異為3.0-5.2%，推測是不同來源的病毒。這是目前首次發現台灣存在兩種不同基因型的第一型鴨A型肝炎病毒，這些發現有助於了解第一型鴨A型肝炎病毒在台灣的演化情形。第三部分:即時定量反轉錄聚合酶鍊反應用於偵測第一型鴨A型肝炎病毒，搭配高解析度融點分析法進一步區分台灣使用的疫苗病毒株5886。這個檢測方法只需要一對引子，操作簡單。引子設計於病毒2A2基因上的高保留區，於目標基因內台灣野外病毒和疫苗病毒的鳥糞嘌呤(G)和胞嘧啶(C)數量不同。野外病毒的GC百分比為41.8%-43.5%高於疫苗病毒株5886 (39.2%)。高解析度融點分析結果顯示，野外病毒半解離溫度為81.6±0.1-82.1±0.1℃高於疫苗病毒的80.3±0.1℃。結果證實這個新的檢測方法的確可檢測並區分台灣的野外病毒與疫苗病毒。特異性和敏感性測試結果顯示，對第一型鴨A型肝炎病毒有極高的特異性，檢測極限約為100個病毒核酸。一日齡幼鴨人工感染病毒，可於感染後6小時，自糞便中檢測出病毒核酸。這個檢測方法，將可用於台灣鴨場對於第一型鴨A型肝炎病毒的檢測和監控。

Avian influenza and Duck hepatitis are important viral diseases of poultry. They spread rapidly, cause high mortality in poultry and make huge economical loss to poultry industry. They are single-strand RNA viruses that can evolve rapidly in response to specific selective pressures. This study analyzed the sequences from avian influenza and duck hepatitis viruses isolated in Taiwan. The first part of this dissertation reports the hemagglutinin cleavage site (HA CS) sequences and pathogenicity of H5N2 viruses that were isolated from chickens in Taiwan during 2013 to 2015. The first outbreak of H5N2 virus in chickens occurred in Taiwan in 2003. Three types of HA CS sequences, QREKR/GL, QRKKR/GL and QRRKR/GL, were previously reported in Taiwanese H5N2 viruses that were isolated from chickens from 2003 to 2013. Two novel HA CS sequences, QKEKR/GL and KREKREKR/GL, were found in the viruses isolated in 2013 and 2014, and pathogenicity tests showed that the viruses with these novel HA CS sequences are low and high pathogenic viruses, respectively. In contrast, the HA CS sequence QREKR/GL was found in all viruses that were isolated in 2015, and all of these viruses were low pathogenic viruses. To study the mechanism by which Taiwanese LPAI H5N2 viruses evolved into HPAI viruses, a LPAI virus that was isolated in 2003 was serial passages in chicken embryonated eggs. After 10 passages, at least four distinct types of HA CS sequences were found in the allantoic fluid. These results highlight the potential of Taiwanese H5N2 viruses to change their pathogenicity and HA CS sequences via mutations. The mutation caused by nucleotide substitutions, deletions and insertions. These finding were useful for understanding the mechanism of sequence changes at the HA CS and for refining H5N2 virus control measures in Taiwan. The second part of this dissertation reports the sequences of duck hepatitis viruses that were isolated from ducklings in Taiwan during 2010 to 2014. Ten viruses were isolated from 52 duck farms, and the positive rate was 19% (10/52). The 10 isolates are identified as DHAV-1 according to their 5’-UTR nucleotide sequences. Phylogenetic analyses of the complete VP1 amino acid sequence showed that these isolates fell into two distinct genetic groups: TW-I and TW-II. Eight virus that were classified into TW-I group appeared to descend from the strain 03D which was previously isolated in Taiwan in 2003. In contrast, TW-II group including 2 isolates that were more closely related to the viruses which were isolated in China. Pair-wise VP1 amino acid sequence comparison revealed that the differences between the TW-I and TW-II groups were 3.0% to 5.2%. This result indicated that viruses of TW-I and TW-II might have separately evolved for a considerable period of time. To date, this is the first report about the presence of two genotype DHAV-1 in Taiwan. This study may be useful to better understand the epidemiology and evolution of DHAV-1 in Taiwan. The third part of this dissertation reports the development of a real-time RT-PCR to detect DHAV-1 and high resolution melting (HRM) assay to differentiate field viruses from the vaccine strain 5886. The operation of this method is simple because of only a set of primer was used. The primers were designed at the conserved sequences of viral 2A2 gene. Taiwanese field isolates were quite different from the vaccine strain 5886 in the G (guanine)-C (cytosine) contents of the target gene. The GC-content percentage of field isolates (41.8-43.5%) were higher than that of the vaccine strain 5886 (39.2%). HRM analyses showed that Tm value of the field isolates (81.6±0.1-82.1±0.1℃) is higher than the vaccine strain 5886 (80.3±0.1℃). This result indicated that the vaccine strain 5886 and the Taiwanese filed strains could be detected and differentiated from each other by the real-time RT-PCR and HRM assay. Specificity and sensitivity tests showed that this assay was highly specific for DHAV-1, and the detection limit was about 100 copies of the viral RNA. Experiments using fecal samples collected from ducklings artificially infected with DHAV-1 showed that viral RNA could be detected in fecal samples as early as 6 hours post infection. In summary, the real-time RT-PCR and HRM assay was developed in this study, and this assay could be valuable for diagnosis and surveillance of DHAV-1 infection in Taiwan.

摘 要 i
Abstract iii
Chapter 1. Introduction 1
1.1. Avian influenza 1
1.2. Duck hepatitis A virus 2
Chapter 2. Sequence diversity and associated pathogenicity of the hemagglutinin cleavage site of H5N2 avian influenza viruses isolated from chickens in Taiwan during 2013-2015 6
2.1. Materials and methods 6
2.1.1. Viruses and isolation of viral RNA 6
2.1.2. Reverse transcription-polymerase chain reaction (RT-PCR) amplification 7
2.1.3. Direct sequencing of the RT-PCR products 7
2.1.4. Sequencing of the cloned RT-PCR products 8
2.1.5. Pathogenicity study 8
2.2. Results 9
2.2.1. RT-PCR amplification of the 194-bp DNA fragment containing the HA CS sequence 9
2.2.2. Sequence diversity and pathogenicity of viruses isolated during 2013 to 2015 9
2.2.3. Sequence diversity in strain 1203/03 and its progeny viruses 10
2.2.4. Detection of viral quasispecies with different HA CS sequences 11
2.3. Discussion 12
Chapter 3. Genetic characterization of duck hepatitis A viruses isolated in Taiwan during 2010 to 2014 14
3.1. Materials and methods 14
3.1.1. Sample collection 14
3.1.2. RNA extraction 14
3.1.3. RT-PCR 14
3.1.4. Virus isolation 15
3.1.5. DNA sequencing 16
3.1.6. Sequence and phylogenetic analyses 16
3.2. Results 16
3.2.1. Virus detection and isolation 16
3.2.2. Sequence analyses of the partial 5’-UTR gene of DHAV 17
3.2.3. Phylogenetic analyses of the VP1 amino acid 17
3.2.4. Identification of a hypervariable region in the VP1 amino acid sequences 18
3.3. Discussion 19
Chapter 4. Detection Taiwanese duck hepatitis A virus type 1 and differentiation from the vaccine strain 5886 using a real-time RT-PCR and high resolution melting assay 22
4.1. Materials and methods 22
4.1.1. Viruses and bacteria 22
4.1.2. Design of real-time RT-PCR primer 22
4.1.3. Extraction of nucleic acid and the Real-time RT-PCR 22
4.1.4. Preparation of standard RNA templates 23
4.1.5. Establishment of a standard curve 23
4.1.6. Specificity of the real-time RT-PCR 24
4.1.7. Reproducibility evaluation 24
4.1.8. A comparative test of DHAV-1 between the conventional RT-PCR and real-time PCR 24
4.1.9. A high resolution melting (HRM) analysis to differentiate between filed isolates and vaccine strain of DHAV-1 25
4.1.10. Sequencing analysis 25
4.2. Results 26
4.2.1. Primer design 26
4.2.2. Standard curve 26
4.2.3. Sensitivity of the real-time RT-PCR 26
4.2.4. Specificity of the real-time RT-PCR 27
4.2.5. Reproducibility 27
4.2.6. HRM analyses 27
4.2.7. Rapid identification of DHAV-1 isolates using the real-time RT-PCR and HRM assay 27
4.2.8. Sequence analyses of the partial 2A2 gene 28
4.2.9. Detection of DHAV-1 in fecal samples 28
4.3. Discussion 29
Chapter 5. Conclusions 31
References 52

鄭明珠, 李敏旭, 李淑慧 & 王金和 (2011) 14日齡雞胚胎累代感染造成之H5N2 低病原性家禽流行性感冒病毒毒力變異. 臺灣獸醫學雜誌, 37, 221-226.

Alexander, D.J. (1995) The epidemiology and control of avian influenza and Newcastle disease. Journal of comparative pathology, 112, 105-126.

Anchun, C., Mingshu, W., Hongyi, X., Dekang, Z., Xinran, L., Haijuen, C., Renyong, J. & Miao, Y. (2009) Development and application of a reverse transcriptase polymerase chain reaction to detect Chinese isolates of duck hepatitis virus type 1. Journal of microbiological methods, 76, 1-5.

Banks, J., Speidel, E.S., Moore, E., Plowright, L., Piccirillo, A., Capua, I., Cordioli, P., Fioretti, A. & Alexander, D.J. (2001) Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy. Archives of virology, 146, 963-973.

Cao, J., Ou, X., Zhu, D., Ma, G., Cheng, A., Wang, M., Chen, S., Jia, R., Liu, M., Sun, K., Yang, Q., Wu, Y. & Chen, X. (2016) The 2A2 protein of duck hepatitis A virus type 1 induces apoptosis in primary cell culture. Virus genes, 52, 780-788.

Cheng, M.C., Soda, K., Lee, M.S., Lee, S.H., Sakoda, Y., Kida, H. & Wang, C.H. (2010) Isolation and characterization of potentially pathogenic H5N2 influenza virus from a chicken in Taiwan in 2008. Avian diseases, 54, 885-893.

Crighton, G.W. & Woolcock, P.R. (1978) Active immunisation of ducklings against duck virus hepatitis. The veterinary record, 102, 358-361.

Curd, E., Pollinger, J., Toffelmier, E. & Smith, T. (2011) Rapid influenza A detection and quantitation in birds using a one-step real-time reverse transcriptase PCR and high resolution melting. Journal of virological methods, 176, 125-130.

Donnelly, M.L., Gani, D., Flint, M., Monaghan, S. & Ryan, M.D. (1997) The cleavage activities of aphthovirus and cardiovirus 2A proteins. The journal of general virology, 78 (Pt 1), 13-21.

Duffy, S., Shackelton, L.A. & Holmes, E.C. (2008) Rates of evolutionary change in viruses: patterns and determinants. Nature reviews genetics, 9, 267-276.

Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., Rimmelzwaan, G.F., Olsen, B. & Osterhaus, A.D. (2005) Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. Journal of virology, 79, 2814-2822.

Fry, E.E., Knowles, N.J., Newman, J.W., Wilsden, G., Rao, Z., King, A.M. & Stuart, D.I. (2003) Crystal structure of Swine vesicular disease virus and implications for host adaptation. Journal of virology, 77, 5475-5486.

Fu, Y., Pan, M., Wang, X., Xu, Y., Yang, H. & Zhang, D. (2008) Molecular detection and typing of duck hepatitis A virus directly from clinical specimens. Veterinary microbiology, 131, 247-257.

Ghorashi, S.A., O'Rourke, D., Ignjatovic, J. & Noormohammadi, A.H. (2011) Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis. Journal of virological methods, 171, 264-271.

HaanWoo, S., JaeHong, K., ChangSeon, S., MyungGuk, H., YounJeong, L., InPil, M. & KiSeuk, K. (2000) Development of a live vaccine strain of duck viral hepatitis using a Korean isolate. Korean journal of veterinary research, 40, 110-116.

Horimoto, T., Rivera, E., Pearson, J., Senne, D., Krauss, S., Kawaoka, Y. & Webster, R.G. (1995) Origin and molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in Mexico. Virology, 213, 223-230.

Huang, S.W., Ho, C.F., Chan, K.W., Cheng, M.C., Shien, J.H., Liu, H.J. & Wang, C.Y. (2014) The genotyping of infectious bronchitis virus in Taiwan by a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction. Journal of veterinary diagnostic investigation, 26, 721-733.

Hwang, J. (1965) A chicken-embryo-lethal strain of duck hepatitis v. Avian diseases, 9, 417-422.

Iqbal, M., Reddy, K.B., Brookes, S.M., Essen, S.C., Brown, I.H. & McCauley, J.W. (2014) Virus pathotype and deep sequencing of the HA gene of a low pathogenicity H7N1 avian influenza virus causing mortality in Turkeys. PLoS one, 9, e87076.

Jin, X., Zhang, W., Zhang, W., Gu, C., Cheng, G. & Hu, X. (2008) Identification and molecular analysis of the highly pathogenic duck hepatitis virus type 1 in Hubei province of China. Research veterinary science, 85, 595-598.

Jones, D.T., Taylor, W.R. & Thornton, J.M. (1992) The rapid generation of mutation data matrices from protein sequences. Computer applications in the biosciences: CABIOS, 8, 275-282.

Kim, M.C., Kwon, Y.K., Joh, S.J., Kim, S.J., Tolf, C., Kim, J.H., Sung, H.W., Lindberg, A.M. & Kwon, J.H. (2007a) Recent Korean isolates of duck hepatitis virus reveal the presence of a new geno- and serotype when compared to duck hepatitis virus type 1 type strains. Archives of virology, 152, 2059-2072.

Kim, M.C., Kwon, Y.K., Joh, S.J., Kwon, J.H., Kim, J.H. & Kim, S.J. (2007b) Development of one-step reverse transcriptase-polymerase chain reaction to detect duck hepatitis virus type 1. Avian diseases, 51, 540-545.

Kim, M.C., Kwon, Y.K., Joh, S.J., Lindberg, A.M., Kwon, J.H., Kim, J.H. & Kim, S.J. (2006) Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae. The journal of general virology, 87, 3307-3316.

Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of molecular evolution, 16, 111-120.

Kumar, S., Stecher, G. & Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular biology and evolution, 33, 1870-1874.

Lee, C.C.D., Zhu, H., Huang, P.Y., Peng, L., Chang, Y.C., Yip, C.H., Li, Y.T., Cheung, C.-L., Compans, R. & Yang, C. (2014) The emergence and evolution of avian H5N2 influenza viruses in chickens in Taiwan. Journal of virology, 88, 5677-5686.

Liu, G., Wang, F., Ni, Z., Yun, T., Yu, B., Huang, J. & Chen, J. (2008) Genetic diversity of the VP1 gene of duck hepatitis virus type I (DHV-I) isolates from southeast China is related to isolate attenuation. Virus research, 137, 137-141.

Londt, B.Z., Banks, J. & Alexander, D.J. (2007) Highly pathogenic avian influenza viruses with low virulence for chickens in in vivo tests. Avian pathology, 36, 347-350.

Mateu, M.G. (1995) Antibody recognition of picornaviruses and escape from neutralization: a structural view. Virus research, 38, 1-24.


Oberste, M.S., Maher, K., Kennett, M.L., Campbell, J.J., Carpenter, M.S., Schnurr, D. & Pallansch, M.A. (1999) Molecular epidemiology and genetic diversity of echovirus type 30 (E30): genotypes correlate with temporal dynamics of E30 isolation. Journal of clinical microbiology, 37, 3928-3933.

OIE. (2008) Avian influenza, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees), 6th Edition, vol. 1. Office Intl Des Epizooties, Paris, pp. 465-481.

Pan, M., Yang, X., Zhou, L., Ge, X., Guo, X., Liu, J., Zhang, D. & Yang, H. (2012) Duck hepatitis A virus possesses a distinct type IV internal ribosome entry site element of picornavirus. Journal of virology, 86, 1129-1144.

Reed, G.H., Kent, J.O. & Wittwer, C.T. (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics, 8, 597-608.

Rossmann, M.G. (1989) The canyon hypothesis. Hiding the host cell receptor attachment site on a viral surface from immune surveillance. The journal of biological chemistry, 264, 14587-14590.

Rott, R. (1992) The pathogenic determinant of influenza virus. Veterinary microbiology, 33, 303-310.

Samuilova, O., Krogerus, C., Poyry, T. & Hyypia, T. (2004) Specific interaction between human parechovirus nonstructural 2A protein and viral RNA. The journal of biological chemistry, 279, 37822-37831.

Senne, D.A., Panigrahy, B., Kawaoka, Y., Pearson, J.E., Suss, J., Lipkind, M., Kida, H. & Webster, R.G. (1996) Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential. Avian diseases, 40, 425-437.

Soda, K., Cheng, M.C., Yoshida, H., Endo, M., Lee, S.H., Okamatsu, M., Sakoda, Y., Wang, C.H. & Kida, H. (2011) A low pathogenic H5N2 influenza virus isolated in Taiwan acquired high pathogenicity by consecutive passages in chickens. The journal of veterinary medical science / the Japanese Society of Veterinary Science, 73, 767-772.

Song, C., Wan, H., Yu, S., Han, X., Qiu, X., Hu, Q., Tan, L. & Ding, C. (2012) Rapid detection of duck hepatitis virus type-1 by reverse transcription loop-mediated isothermal amplification. Journal of virological methods, 182, 76-81.

Steer, P.A., Kirkpatrick, N.C., O'Rourke, D. & Noormohammadi, A.H. (2009) Classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region. Journal of clinical microbiology, 47, 311-321.

Tseng, C.H., Knowles, N.J. & Tsai, H.J. (2007) Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new genus. Virus research, 123, 190-203.

Tseng, C.H., Shih, Y.H., Chen, P.W., Jian, L.Q., Huang, T.S. & Huang, C.C. (2009) Characterization and sequence analyses of duck hepatitis viruses isolated in 2008. Exp. Rep. AHRI, 44, 25-35.

Tseng, C.H. & Tsai, H.J. (2007) Molecular characterization of a new serotype of duck hepatitis virus. Virus research, 126, 19-31.

Van den Hoecke, S., Verhelst, J., Vuylsteke, M. & Saelens, X. (2015) Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing. BMC genomics, 16, 79.

Wang, Y., Li, C., Chen, Z., Xu, B., Li, G. & Liu, G. (2012) Complete genome comparison of duck hepatitis virus type 1 parental and attenuated strains. Virus genes, 45, 398-401.

Wen, X.J., Cheng, A.C., Wang, M.S., Jia, R.Y., Zhu, D.K., Chen, S., Liu, M.F., Liu, F. & Chen, X.Y. (2014) Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay. Poultry science, 93, 2184-2192.

WHO. 2014. Cumulative number of confirmed human cases of avian influenza A(H5N1) reported to WHO. http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/. [accessed 15 June, 2017].


Woolcock, P.R. & Tsai, H.J. (2013) Duck hepatitis. In Swayne, D.E., Glisson, J.R., McDougald, L.R., Nolan, L.K., Suarez, D.L., Nair, V.L. (eds) Diseases of Poultry. Wiley-Blackwell, Ames. Iowa, pp. 422-431.

Wu, X., Li, X., Zhang, Q., Wulin, S., Bai, X., Zhang, T., Wang, Y., Liu, M. & Zhang, Y. (2015) Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein. PLoS One, 10, e0118041.

Yang, L., Li, J., Bi, Y., Xu, L. & Liu, W. (2012) Development and application of a reverse transcription loop-mediated isothermal amplification method for rapid detection of duck hepatitis A virus type 1. Virus genes, 45, 585-589.

Yang, M., Cheng, A., Wang, M. & Xing, H. (2008) Development and application of a one-step real-time Taqman RT-PCR assay for detection of Duck hepatitis virus type1. Journal of virological methods, 153, 55-60.