2023-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/654027在病毒B型肝炎慢性感染的期間，病毒體更新快速，突變率高，而且族群規模龐大。因此，在宿主體內積累許多的遺傳變異。揭露病毒基因組突變產生以及頻率增加的過程，有助於吾人瞭解宿主體內疾病進展和病毒演化的過程。為了研究突變以及天擇對於病毒族群演化速率的相對角色，研究者必須了解基本突變的輪廓，即不同種類的突變在基因組不同區域的產生速率。將病毒在宿主體內的遺傳變異與基本突變輪廓比較，其差異即為天擇所造成。本計畫將解析天擇以及突變如何主導B肝病毒在宿主體內的演化，以及它們相對的重要性。首先，我們以獨特分子標記建立一個利用第三代包含Oxford Nanopore or Pacific Biotech定序方式，得到高正確性長序列的流程。利用這個流程，我們將分析B肝病毒中由宿主RNA聚合酶 II以及病毒反轉錄酶在細胞培養下所產生的突變，並以此系統分析不同B肝病毒基因型(A, B, C, D)突變之異同。接著我們也會研究B肝病毒轉殖小鼠中病毒的突變。這些B肝病毒在細胞株以及小鼠體內的突變，將會與從人類宿主身上所產生的突變比較。他們的差異可以讓我們了解B肝病毒在慢性感染中受到天擇影響的程度。 During chronic HBV infection, and in other virus infections as well, the turnover of virions is rapid, the mutation rate is high, and the population size is large. Consequently, a huge amount of genetic variation is accumulated within hosts. This varia-tion allows viruses to adapt to the immune system and to acquire drug resistances. The ge-netic variation of viruses is shaped by both mutations and natural selection. Revealing the processes that govern the rate of mutation generation and strength of selection is therefore central to our understanding of disease progression and virus evolution within hosts. To get the relative contribution of selection and mutation in shaping the tempo of nucleotide change in viral population, one first has to estimate the underlying mutation profile, i.e., rate and pattern of mutation generated in different parts of the genome. By subtracting this mu-tation profile from genetic variation derived from populations, the real effect of natural se-lection can be revealed. The goal of this study is to dissect the relative contribution of mu-tation and selection in shaping HBV dynamics within hosts. To that end, we will first estab-lish and optimize a pipeline which can generate high accuracy long read amplicon sequence using universal molecular identifiers (UMIs) with 3rd generation Oxford Nanopore or Pa-cific Biotech sequencing. Using this pipeline, we aim to examine mutations in RC- DNA representing errors introduced mainly by host polymerase II and virus polymerase in cell culture system (in vitro) for different HBV genotypes, i.e., genotype A, B, C, and D. Fi-nally, the underlying mutation profiles of HBV in vivo will be examined using hydrody-namic injection mouse model. Comparing HBV mutation profiles from in vivo and in vitro assays with HBV genetic variation from humans, we will get the correct estimates of selec-tion force on the virus during chronic infection.B肝病毒;天擇;獨特分子標記;突變率;遺傳變異;hepatitis B virus; natural selection; universal molecular identifiers (UMIs); mutation rate; genetic variation