蔡向榮臺灣大學：獸醫學研究所曾俊憲Tseng, Chun-HsienChun-HsienTseng2007-11-282018-07-092007-11-282018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/60001There were two epidemics of duck viral hepatitis (DH) in 1971 and 1990 in Taiwan, respectively, and both epidemics caused massive deaths in ducklings. During the first epidemic immune sera collected from the infected and survived ducks were used to control the disease, and then a vaccine based on the 5886 strain duck hepatitis virus type 1 (DHV-1) was developed and used in the field. Since then the DHV-1 vaccine was used to vaccinate breeder ducks, and yolk antibody against DHV-1 was also used in day-old ducklings in most of the duck farms in Taiwan. The second epidemic of DH occurred in 1990, with the co-infection of waterfowl parvovirus and led to the death of a toll of 300,000 ducklings. It was suggested by some researchers that the inadequacy practice of DHV-1 vaccine maybe the cause of the epidemic of DH in 1990. After 1990, the DHV-1 vaccine and yolk antibody have remained the two important tools for the prevention and control of the DH in duck farms. But sometimes disputes arise over some of the one-week old ducklings still suffered from DH even the DHV-1 vaccine and yolk antibody have been applied in the farms. The DHV-1 was fist outbreak in Long Island, USA, about a half century ago, but there still had no any nucleic acid sequence been published. Thus the diagnosis of DHV-1 infection still relied on the traditional virus isolation and identification methods or serology assay. In order to realize the real cause of the DH in the field and also to develop the molecular diagnostic method for DH, total of six strains of duck-originated picornavirus were characterized, sequenced and pylogenetic analyzed in this study. In this study, we have successfully decoded the complete genome sequences of six trains of duck-originated picornavirus and revealed the existence of three types of duck-originated picornaviruses, Duck hepatitis virus type 1 (DHV-1), new serotype duck hepatitis virus (N-DHV) and Duck picornavirus (DPV). After comparing these genome sequences with other picornaviruses, it has revealed that although all these 3 duck-originated picornaviruses belong to Picornaviridae, their genome structures, sequence similarities and evolution analyses are quite different from other known picornaviruses. Therefore, in the future DHV-1 and N-DHV will probably be classified into a new genus, Avihepatovirus, and DPV with several serotypes of simian picornavirus and serotype 8 of porcine enterovirus will be classified into another new genus, Sapelovirus. The reasons for theses 3 duck-originated picornaviruses will probably be classified into two new genera are based on the 3 duck-originated picornaviruses possess complete different genome structures and the similarities of their polyprotein sequences are less than 30% comparing to other known picornaviruses. The major difference between DHVs and other picornaviruses is that genome of DHVs possess three in-tandem 2A genes. 2A1, 2A2, and 2A3 proteins, represented an aphthovirus-like 2A protein, AIG1-like protein, and human parechovirus-like 2A protein, respectively. And the pair-wise amino acid sequence identities between polyprotein of DHVs and other picornaviruses are all less than 30%. The pair-wise amino acid sequence identities in the 3D region of DHVs with ljungan virus and human parechovirus type 1 is only 38.6% and 36.6%, respectively, and less than 30% with all other picornaviruses. As to DPV, its genome possess several different characteristics from other picornaviruses, i.e. the L protein, composed of 451 amino acids, is the largest within the family Picornaviridae, the 2A protein was composed of only 12 amino acids, which is the shortest of any member of the family Picornaviridae, and the phylogenetic analysis of the polyprotein and 3D sequences indicated that the DPV together with the porcine enterovirus type 8 virus and several simian picornaviruses form a distinct branch of the family Picornaviridae. In this study, we first demonstrated the existence of 3 types of duck-originated picornaviruses in Taiwan. The two serotypes of duck hepatitis viruses, DHV-1 and N-DHV, are highly virulent to ducklings under 3-week old. The mortality was above 80% in ducklings infected with either DHV-1 or N-DHV, and it was indistinguishable between DHV-1 and N-DHV by the postmortem and pathological examinations, and the mortality rates. It is suspected that the outbreaks of DH in the duck farms that DHV-1 vaccine and yolk antibody have been used might be due to the infection of the N-DHV. With the complete decoded of the genome sequences, it will be very easy to differentiate between DHV-1 and N-DHV infection in the field by the molecular diagnostic method. As to DPV, its virulence was much lower than DHVs and didn’t cause high mortality in infected duck flocks. Only one-day-old ducklings suffered from DPV could lead to growth inhibition and result in an economic loss in the infected duck farms.Certificate I Acknowledgments II Abstract III Abstract in Chinese VI Contents VIII Chapter 1. Introduction 1 Chapter 2. Literature Review 4 Chapter 3. Molecular analysis of duck hepatitis virus type 1 indicates that it should be assigned to a new Picornaviridae genus 13 3.1. Abstract 13 3.2. Abstract in Chinese 13 3.3. Introduction 14 3.4. Materials and methods 16 3.4.1. Viruses and cells 16 3.4.2. Neutralization assays 17 3.4.3. Isolation and purification of DHV-1 RNA 18 3.4.4. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of DHV-1 18 3.4.5. Sequence analysis 19 3.4.6. Accession numbers of the sequences used in this study 20 3.5. Results and discussion 21 3.5.1. Neutralization assays 21 3.5.2. Sequencing genomes of three DHV-1 isolates 21 3.5.3. Features of the DHV-1 genome and deduced polyprotein sequence 22 3.5.4. 5’ UTR 25 3.5.5. 2A1 26 3.5.6. 2A2 27 3.5.7. 2A3 28 3.5.8. 3’ UTR 29 3.5.9. Structure proteins 29 3.5.10. Non-structural proteins 31 3.5.11. Phylogenetic analyses 32 3.6. Conclusion 32 Chapter 4. Molecular characterization of a new serotype of duck hepatitis virus 34 4.1. Abstract 34 4.2. Abstract in Chinese 34 4.3. Introduction 35 4.4. Materials and methods 36 4.4.1. Isolation of N-DHV and preparation of antiserum 36 4.4.2. Physicochemical characterization 37 4.4.3. Cross-neutralization tests 38 4.4.4. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of N-DHV 38 4.4.5. Sequence analysis 39 4.5. Results and Discussion 40 4.5.1. Physicochemical characterization 40 4.5.2. Cross-neutralization assay 40 4.5.3. Features of the N-DHV genome and deduced polyprotein sequence 41 4.5.4. 5’UTR 43 4.5.5. 2A 44 4.5.6. 3’UTR 45 4.5.7. Structural proteins 45 4.5.8. Non-structural proteins 48 4.5.9. Phylogenetic analyses 49 4.6. Conclusion 49 Chapter 5. Sequence analysis of a duck picornavirus isolate indicates that it together with porcine enterovirus type 8 and simian picornavirus type 2 should be assigned to a new picornavirus genus 51 5.1. Abstract 51 5.2. Abstract in Chinese 52 5.3. Introduction 53 5.4. Materials and methods 55 5.4.1. Virus growth and characterization 55 5.4.2. Pathogenicity test 55 5.4.3. Production of the hyperimmune sera 56 5.4.4. Cross-neutralization tests 56 5.4.5. Purification of duck picornavirus TW90A 56 5.4.6. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of DPV 57 5.4.7. Sequence analysis 57 5.4.8. Protein sequencing 58 5.5. Results and Discussion 59 5.5.1. Virus growth and characterization 59 5.5.2. Pathogenicity test 60 5.5.3. Cross-neutralization tests 60 5.5.4. Features of the DPV genome and deduced polyprotein sequence 61 5.5.5. 5’UTR 63 5.5.6. 3’ UTR 65 5.5.7. Leader protein of the DPV 65 5.5.8. Structural and non-structural proteins of the DPV 66 5.5.9. Phylogenetic analysis 67 5.5.10. Protein sequencing 68 5.6. Conclusion 69 Chapter 6. Conclusions 71 Figures 80 Tables 99 References 112 Author introduction 1241112196 bytesapplication/pdfen-US小核糖核酸病毒鴨肝炎病毒picornavirusduck hepatitis virus[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/60001/1/ntu-96-D89629002-1.pdf