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Study on Molecular Genetic Analysis of Bovine Leukemia Virus in South America and Asia

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Study on Molecular Genetic Analysis of Bovine Leukemia Virus in South America and Asia 博士論文 Study on molecular genetic analysis of bovine leukemia virus in South America and Asia (南米とアジアにおける牛白血病ウイルスの分子遺伝学的解析) 买热帕提 帕拉提 Mairepati Palati Contents Abbreviations ........................................................................................................... 4 Abstract ..................................................................................................................... 6 General introduction..............................................................................................11 CHAPTER I. ............................................................................................................... Detection and molecular characterization of bovine leukemia virus in Philippine cattle ...18 1-1. Introduction ........................................................................................................................ 19 1-2. Materials and Methods ....................................................................................................... 22 1-2-1 Experimental samples ........................................................................................................... 22 1-2-2 Animal handling and research Ethics .................................................................................. 22 1-2-3 Genomic DNA extraction methods ..................................................................................... 22 1-2-4 Detection of BLV provirus ................................................................................................... 23 1-2-5 PCR amplification and sequencing of BLV env-gp51 gene fragments .......................... 24 1-2-6 Construction of a phylogenetic tree ..................................................................................... 25 1-3. Results ................................................................................................................................ 26 1-3-1 Detection of BLV infection by BLV-CoCoMo-qPCR-2 and nested PCR ..................... 26 1-3-2 Phylogenetic analysis based partial sequence of BLV env gp51 region ........................ 26 1-3-3 Nucleotide and amino acid substitutions of BLV env-gp51 from the Philippines strains ......... 27 1-4. Discussion .......................................................................................................................... 29 1-5. Conclusions ........................................................................................................................ 32 CHAPTER II. ............................................................................................................. A new genotype of bovine leukemia virus in South America identified by NGS-based whole genome sequencing and molecular evolutionary genetic analysis ....................................33 2-1. Introduction ........................................................................................................................ 34 2-2. Materials and Methods ....................................................................................................... 37 2-2-1 Experimental samples ........................................................................................................... 37 2-2-2 Animal handling and research Ethics .................................................................................. 37 1 2-2-3 Genomic DNA extraction methods ..................................................................................... 37 2-2-4 Detection of BLV provirus by nested PCR targeting BLV-LTR gene ........................... 37 2-2-5 PCR amplification and sequencing of BLV env-gp51 gene fragments .......................... 38 2-2-6 Construction of a phylogenetic tree based on gp51 partial sequences............................ 39 2-2-7 PCR amplification of the BLV provirus complete genome ............................................. 40 2-2-8 BLV whole genome library preparation ............................................................................. 41 2-2-9 Next-generation sequencing (NGS) data analysis ............................................................. 41 2-3. Results ................................................................................................................................ 43 2-3-1 Investigation of the prevalence in South America............................................................. 43 2-3-2 Identification of a novel BLV genotype, genotype-9, by phylogenetic analysis based on partial env gp51 sequences ........................................................................................................ 44 2-3-3 Whole genome sequencing and sequence comparison of strains of genotypes-1, -2, and -6, and novel genotype-9 .......................................................................................................... 45 2-3-4 Confirmation of the existence of novel BLV genotype, genotype-9 through phylogenetic analysis based on whole genome sequences ......................................................... 46 2-3-5 Diversity of structural and non-structural genes of 16 complete genome sequence of novel BLV strains from Peru, Paraguay and Bolivia in South America ................................... 46 2-3-6 Observation of novel genotype-9 specific mutations ........................................................ 51 2-4. Discussion .......................................................................................................................... 53 2-5 Conclusion .......................................................................................................................... 56 CHAPTER III. ........................................................................................................... Confirmation of genotype-10 of bovine leukemia virus in Myanmar by full genome sequences ....58 3-1. Introduction ........................................................................................................................ 59 3-2. Materials and Methods ....................................................................................................... 61 3-2-1 Experimental samples ........................................................................................................... 61 3-2-2 Animal handling and research Ethics .................................................................................. 61 3-2-3 Genomic DNA extraction methods ..................................................................................... 61 3-2-4 Detection of BLV provirus ................................................................................................... 62 3-2-5 PCR amplification and sequencing of BLV env-gp51 gene ............................................ 63 3-2-6 PCR amplification of the BLV provirus complete genome ............................................. 64 2 3-2-7 Cloning of the PCR products for sequencing of BLV full genome ................................ 65 3-3. Results ................................................................................................................................ 66 3-3-1 Investigation of the spread of BLV infection in Myanmar .............................................. 66 3-3-2 Phylogenetic analysis of BLV strains based on env-gp51 partial fragment and full region ................................................................................................................................................. 66 3-3-3 Nucleotide sequence analysis of partial env gp51 region of BLV strains in Myanmar ........... 67 3-3-4 Amino acid sequence analysis of partial gp51 Env protein of BLV strains in Myanmar ......... 68 3-3-5 Maximum-likelihood phylogenetic tree based on BLV full genome sequences ........... 69 3-3-6 Amino acid comparison of each BLV gene among Myanmar strains and other known strains ....... 69 3-4 Discussion ........................................................................................................................... 71 3-5. Conclusions ........................................................................................................................ 74 General Discussion .................................................................................................75 AKNOWLEDGEMENT ........................................................................................78 References ...............................................................................................................80 Tables ......................................................................................................................96 Figures and Figure legends .................................................................................110 3 Abbreviations EBL : Enzootic bovine leukosis SBL : Sporadic bovine leukosis BLV : Bovine leukemia virus PL : Persistent lymphocytosis STLV : Simian T-lymphotropic virus HTLV : Human T-cell leukemia virus nt : Nucleotide LTR : Long terminal repeats CA : Capsid protein NC : Nucleocapsid protein MA : Matrix protein RNA : Ribonucleic acid ENV : Envelop protein IN : Integrase RT : Reverse transcriptase SU : Surface unit TM : Transmembrane protein ITAM : Immunoreceptor tyrosine-based activation TxRE : Tax-responsive element CRE : Cycle-AMP responsive element CREB : Cycle-AMP responsive element binding protein REF : Rat embryo fibroblasts Ha-ras : Hayvey rat sarcoma viral oncoprotein mRNA : Messenger RNA miRNA : MicroRNA 4 DNA : Deoxyribonucleic acid dsDNA : Double strand DNA cDNA : Complementary DNA PCR : Polymerase chain reaction AGID
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