在單鹼基解析度下探索B型肝炎病毒複製錯誤率

Abstract

基因多樣性源自於突變，並且在演化裡扮演着重要的角色。儘管B型肝炎病毒是一種DNA病毒，但是它在複製過程中會使用到自己的反轉錄酶，使其演化速率趨近於RNA病毒。雖然目前有許多B型肝炎病毒的相關研究，可是我們對其突變特徵仍然不明，局限了我們對於B型肝炎病毒基因多樣性的認知。本研究透過在每個分子上標記獨特辨識碼，達成單一分子解析度，同時利用第三代定序技術，如Oxford Nanopore Technology (ONT) 與 Pacific Biosciences (PacBio)，對在 in vitro中只有複製過一次的病毒進行接近全基因的定序。本研究在橫跨B型肝炎病毒98%的基因組進行定序，ONT與PacBio個別發掘了8,500 和6,300 個突變，並達到大於80,000的定序深度。從定序結果裡，根據兩個不同的定序平台裡估算出B型肝炎病毒在每個位點每次複製之錯誤率為2.28 × 10-5 (PacBio)及3.28 × 10-5 (ONT) 個核苷酸，與C型肝炎病毒相似。兩個定序平台算出的突變差異分別為162倍(ONT)及29倍(PacBio)。在N/S比例裡發現，實際人類族群裡的B型肝炎病毒個別基因的N/S比例為0.18至1.15，但是本研究的in vitro裡發現個別基因的N/S比例為2.65至3.04，姑且推估在人體裡有52到94%的非同義突變是有害並且會被移除。除此之外，本研究找到十九個B型肝炎病毒的剪接變異體，其中五個為新型剪接變異體。以我們所知，本研究是第一個成功估算B型肝炎病毒之單次複製錯誤率、在一個無篩選壓力的環境裡觀察此病毒的突變模式及透過單一分子解析度定序98%基因組探索此病毒之突變特徵。本研究可作為B型肝炎病毒在自然選汰、基因負荷及可演化性裡的基礎狀態，從此可以對其抗藥性及免疫逃避風險提供資料。

Spontaneous mutations, serving as the ultimate source of genetic variation, play a prominent role in evolution. Hepatitis B virus (HBV), while being a DNA virus, utilises its own error-prone reverse transcriptase for replication, giving it an evolutionary rate closer to that of an RNA virus. Although HBV is a well-studied virus, its nucleotide substitution profile remains poorly understood, which limits our comprehension of how HBV generates diversity. This study achieved single-molecule resolution by tagging each molecule with unique molecular identifiers and utilised third-generation sequencing techniques, including Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio), to sequence HBV genome that had only undergone a single round of replication in vitro. We identified 8,500 and 6,300 mutations from ONT and PacBio, respectively, spanning across 98% of the HBV genome, with an average depth of >80,000. The estimated replication error rates of HBV from PacBio and ONT are 2.28 × 10-5 and 3.28 × 10-5 nucleotide/site/replication, respectively, which is the same as HCV. The differences in mutability was found to be 162-fold and 29-fold for ONT and PacBio, respectively. In contrast to inter-hosts studies, where the N/S ratio for different genes ranged between 0.18 and 1.15, the in vitro investigation from this study shows a ratio ranging from 2.65 to 3.04, suggesting that approximately 52 to 94% of nonsynonymous mutations generated within hosts were deleterious and subsequently removed. Furthermore, we have identified nineteen HBV splice variants, five of which are novel. To our knowledge, this is the first study to estimate the error rate of HBV on a per replication basis, demonstrate the mutation pattern of HBV in a selectively neutral environment, and explore the nucleotide substitution profile of nearly full-length HBV with single-molecule resolution. These findings establish a site-specific reference for scrutinizing the aspects of natural selection, genetic load, and HBV's evolutionary potential. Such insights are instrumental in evaluating the imminent risks tied to drug resistance and immune evasion.