e抗原陽性B型肝炎帶原者病毒基因體之演化研究: 著重於不同臨床階段基因型B與C之比較

Abstract

B型肝炎病毒在台灣成人當中有15-20%的帶原率，大部份是由垂直感染而來，其自然病史的分期，可以依據病毒和宿主之間的交互作用，分為四期: immune tolerance phase、immune clearance phase、low replication phase和reactivation phase。相同地，影響B型肝炎病程的因素有很多，包括宿主、病毒本身以及其他環境因素。針對HBV病毒因子來看，不同的genotype在臨床上有不同的表現。台灣的慢性B型肝炎帶原者大多數為genotype B和C，整體而言，genotype B的預後比genotype C好，genotype B比genotype C較早產生eAg seroconversion。若使用血清ALT值作為肝發炎指標來觀察，當病程由immune tolerance phase 進入immune clearance phase時，ALT值會由正常的情況開始逐漸上升或者有數次的波動，而在整個過程當中genotype C有比較長的immune clearance period。不同genotype的HBV genome演化速度與病程發展也許有關，我們因此假設病毒genome的演化速度在immune clearance phase會高於immune tolerance phase，而genotype C的病毒genome演化速度會比genotype B快。 爲釐清這些議題，我們選取24位e抗原陽性之HBV帶原者並且持續追蹤其血清ALT值，並依血清ALT值分成2個groups : group I為 ALT值持續正常者，group II為ALT值有異常升高者。每一個病人在追蹤前後有兩個抽血點分析其HBV全長的基因體序列，時間相隔3年左右。我們萃取DNA並且定量分型HBV，再利用PCR產物直接定序HBV全長的基因體序列，接著使用DNaSP軟體算出此二個抽血點間的HBV演化速度，並且使用合適的統計方法分析這些演化速度在不同genotype及在不同ALT值下有無差異性。由於HBV基因體構造的特殊性，我們分別針對full genome (Pi)、non-overlapping region的synonymous (Ks)和 non-synonymous (Ka)、以及overlapping region (K)分析四種演化速度，。 經排除有pre-S deletion的genotype C序列後，我們計算每一個病人兩個抽血點間的HBV演化速度。我們發現在immune clearance phase的演化速度會大於在immune tolerance phase的演化速度 (Pi、Ks、Ka及K，p值皆小於0.01)。genotype B與genotype C病毒genome的演化速度並無明顯差異，但若以追蹤時間為橫軸，追蹤ALT值為縱軸，算出ALT相對時間的面積，除以時間後得到ALT的平均值，接著計算Ka/yr與平均值的關係，則發現genotype B的病毒genome演化速度與平均時間ALT值有正相關(R2 = 0.5856, p＜0.001 )，而genotype C沒有此一相關性(R2 = 0.0127, p＞0.05 )。 病毒的演化速度在immune clearance phase比在immune tolerance phase快，此結果與我們原先的假設相符。genotype B與genotype C病毒genome的演化速度並無明顯差異；然而考慮ALT值的影響後，發現genotype B的病毒genome演化速度與平均時間ALT值有正相關，genotype C卻無此相關，這樣的結果與我們原先的假設不一致，我們推論genotype B的病毒genome可能對於宿主的免疫反應較genotype C敏感。最後我們並未探討pre-S deletion，因此可能低估genotype C的演化速度。綜合以上，我們推論宿主的免疫篩選在病毒的演化過程中扮演相當重要的角色。

Patients with genotype HBV B infection have lower serum ALT level, and earlier HBeAg seroconversion than genotype C. Whether this different manifestation is related to the HBV genomic evolution remains unknown. We thus investigated the HBV genomic evolution rate in patients at different stage of HBeAg-positive HBV infection We enrolled 24 HBeAg-positive patients (genotype B in 12 and genotype C in 12) with a mean follow-up period of 3 years: 7 with persistently normal serum ALT level (group I, immune tolerance phase), and 17 with abnormal serum ALT level (group II, immune clearance phase). The evolution rate of paired full-length viral genome at enrollment and at last follow-up of each patient was compared according to HBV genotype and serum ALT level by DNaSP 5.0 software and Mann-Whitney test. We counted the evolution rate in full genome (Pi), non-overlapping region [as synonymous (Ks) and non-synonymous (Ka)], and overlapping region (K), respectively. The evolution rate was low in group I patients (1.35×10-5±3.58×10-5 nucleotide substitution/site/year), and significantly increased in group II (1.54×10-3±.1.8×10-3, p<0.001). In general, the evolution rate was similar between genotype B and C in immune tolerance phase or clearance phase. However, a positive correlation between Ka/yr and the area under the average ALT-time curve was found in genotype B (R2 = 0.5856, p＜0.001 ), but not in genotype C (R2 = 0.0127, p＞0.05 ). As anticipated, the HBV genomic evolution rate in immune clearance phase tended to be higher than that in immune tolerance phase. However, in contrast to our speculation, the genomic evolution rate was higher in genotype B than in genotype C. We speculated that HBV genotype B may be more sensitive to host immune response. To be noted, we excluded the genotype C with pre-S deletion from statistical analysis, Thus, the evolution rate of genotype C may be underestimated. Based on these findings, we suggest that immune selection seemed to play a critical role in HBV genome evolution.