利用高通量次世代定序探討在干擾素誘發之E抗原陰轉過程中B型肝炎病毒之演化

Abstract

慢性B肝的疾病表徵較為多變，從非活性帶原者到產生併發症，肝硬化甚至肝癌均可發生。B型肝炎的自然感染史，主要分成三個階段包括免疫耐受期、免疫廓清期以及殘餘期。由免疫耐受期進入非活性帶原期時e抗原消失、e抗體出現，此稱為e抗原消失/陰轉，為慢性B型肝炎進程的主要指標，此時期B型肝炎病毒複製量降低以及肝組織受傷情況緩解。e抗原消失/陰轉也可作為e 抗原陽性之慢性B肝患者停止治療的指標。由之前的研究，發現到某些特定的突變在e抗原消失/陰轉時期會發生例如位於Precore基因位於1896的G變A點突變或是Basal core promoter位於1762以及1764位置的A變T與G變A的突變。在這些突變發生的過程病毒與宿主間的交互作用究竟扮演了怎麼樣的角色，目前還並未有明確的答案。有研究蒐集了e抗原陰轉時期的前後病毒進行序列分析並試著用quasispecies的概念解釋體內病毒的演化，其結果發現在e抗原陰轉發生的前後病毒的歧異度會逐漸上升尤其以有發生e抗原陰轉的族群較為明顯，也許與病毒要對抗免疫反應所產生的適應現象。 之前研究B 型肝炎病毒基因體的變化通常是藉由直接定序與序列選殖的方式進行，此類方式其得到的結果不足以代表整體之情況或是需耗費大量勞力。利用高通量次世代定序理論上可以在一次的定序中得到病毒群體中的每個病毒DNA，較適合進行病毒演化之分析。本實驗利用高通量次世代定序的方法，針對干擾素治療的慢性B肝患者其血清中HBV DNA進行定序，並配合治療時程的不同時間點觀察e抗原消失/陰轉前後病毒DNA序列的演化。由實驗結果顯示在四個主要的基因片段，病毒的序列歧異度均會隨著治療的時間逐漸上升，此現象在有e抗原陰轉的族群中較為顯著，而沒有e抗原陰轉的族群雖然上升的趨勢但較不明顯。另外，將整段HBV DNA去計算其核甘酸最大改變量時，可以發現在三個地方有較明顯的變化。分別是Precore/core、Pre-S以及DR2至basal core promoter的區域。這三段的變化在有發生e抗原陰轉的族群中，可以觀察到較多變化量大於50%的核甘酸，這也許顯示了免疫反應參與其中。但究竟是否為synonymous mutation 或是nonsynonymous mutation 仍需分析此序列點突變造成的胺基酸變異，是否會使病毒產生改變進而影響病毒對抗宿主免疫系統或是躲避免疫系統的攻擊。 總而言之，e抗原陰轉對於慢性B型肝炎來說，一個重要的進程。不僅會影響後續產生其他病症的可能性，也是對於給予藥物治療時間長度的一個參考指標。藉由研究e抗原陰轉中病毒序列的改變，並了解與宿主的交互作用也許可以對目前發生在e抗原陰轉現象提供一個合理的解釋。並對之後藥物的研發也會有所貢獻。

The disease spectrum of chronic hepatitis B (CHB) varies widely, ranging from an inactive carrier state to severe hepatic complications, such as liver cirrhosis and even hepatocellular carcinoma (HCC). The natural history of CHB is characterized by three distinctive phases of disease, including the immune tolerance, immune active, and residual phases. HBeAg loss/seroconversion is a landmark in the natural course of CHB, signifying the transition from an immune active phase to an inactive carrier state. It is often accompanied by the reduction of HBV replication and remission of hepatic injury. HBeAg seroconversion also serves a surrogate therapeutic endpoint for HBeAg-positive CHB patients. The most common precore mutant is the point mutation of nucleotide 1896 of HBV genome (G1896A). The basal core promoter variant encompassing the dual mutationsA1762T and G1764A are also common in HBV mutants. However, the interaction between virus and host does not have a clear answer. Previous studies of HBV quasispecies during HBeAg seroconversion by Lim and colleagues observed that sequence diversity in nonseroconverters was persistently lower compared to seroconverters. In order to answer the phenomenon in more details, we developed a method that take advantage of the next-generation sequencing (NGS) to obtain the massive sequences. The method can obtain a much larger number of sequences than clonal sequencing and give us more information. The result showed that both seroconverters and non-seroconverters had a very similar level of genetic diversity at baseline. Along with the treatment course, the genetic diversity rose in the precore/core, pre-S, polymerase and X regions in seroconverters. However, non-seroconvertes also showed a little high level of genetic diversity at the end of treatment and 6 months after treatment. We also calculated the nucleotide frequency change rates, and found that the rates were higher in precore/core, pre-S and the promoter region located between DR2 to the basal core promoter. In order to identify whether the changes cause synonymous or nonsynonymous mutations, the viral amino acid sequences have to be analyzed. By understanding the virus sequence evolution during HBeAg seroconversion, we are able to gain insight into the pathogenesis of chronic HBV infection, and how the virus adapts and evades the host immune attack. Finally, it may provide new therapeutic approaches for improving the care of CHB patients.