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