Analysis of Precore/Core Covariances Associated with Viral Kinetics and Genotypes in Hepatitis B e Antigen-Positive Chronic Hepatitis B Patients

Hepatitis B virus (HBV) is one of the most common DNA viruses that can cause aggressive hepatitis, cirrhosis and hepatocellular carcinoma. Although many people are persistently infected with HBV, the kinetics in serum levels of viral loads and the host immune responses vary from person to person. HBV precore/core open reading frame (ORF) encoding proteins, hepatitis B e antigen (HBeAg) and core antigen (HBcAg), are two indicators of active viral replication. The aim of this study was to discover a variety of amino acid covariances in responses to viral kinetics, seroconversion and genotypes during the course of HBV infection. A one year follow-up study was conducted with a total number of 1,694 clones from 23 HBeAg-positive chronic hepatitis B patients. Serum alanine aminotransferase, HBV DNA and HBeAg levels were measured monthly as criteria for clustering patients into several different subgroups. Monthly derived multiple precore/core ORFs were directly sequenced and translated into amino acid sequences. For each subgroup, time-dependent covariances were identified from their time-varying sequences over the entire follow-up period. The fluctuating, wavering, HBeAg-nonseroconversion and genotype C subgroups showed greater degrees of covariances than the stationary, declining, HBeAg-seroconversion and genotype B. Referring to literature, mutation hotspots within our identified covariances were associated with the infection process. Remarkably, hotspots were predominant in genotype C. Moreover, covariances were also identified at early stage (spanning from baseline to a peak of serum HBV DNA) in order to determine the intersections with aforementioned time-dependent covariances. Preserved covariances, namely representative covariances, of each subgroup are visually presented using a tree-based structure. Our results suggested that identified covariances were strongly associated with viral kinetics, seroconversion and genotypes. Moreover, representative covariances may benefit clinicians to prescribe a suitable treatment for patients even if they have no obvious symptoms at the early stage of HBV infection.乙型肝炎病毒（Hepatitis B virus, HBV）是最常见的可引发侵袭性肝炎、肝硬化及肝细胞癌的DNA病毒之一。尽管多数人群会持续感染HBV，但病毒载量的血清动力学变化与宿主免疫应答存在显著个体差异。HBV前核心/核心开放读码框（precore/core open reading frame, ORF）编码的乙型肝炎e抗原（hepatitis B e antigen, HBeAg）与核心抗原（core antigen, HBcAg），是病毒活跃复制的两项标志性指标。本研究旨在探究HBV感染进程中，针对病毒动力学、血清转换及基因型的各类氨基酸共变异模式。研究对23例HBeAg阳性慢性乙型肝炎患者开展了为期一年的随访，共获取1694个克隆株。研究人员每月检测血清丙氨酸氨基转移酶、HBV DNA及HBeAg水平，以此为依据将患者划分为若干不同亚组。随后对每月获取的多个前核心/核心ORF进行直接测序，并翻译为氨基酸序列。针对每个亚组，在整个随访周期内，从其时序序列中识别出随时间变化的共变异模式。相较于静止型、下降型、HBeAg血清转换型及基因型B亚组，波动型、迁延型、HBeAg非血清转换型及基因型C亚组的共变异程度更高。结合文献可知，本研究识别出的共变异区域内的突变热点与感染进程密切相关，且此类热点在基因型C中占主导地位。此外，为确定与上述时序共变异模式的交集，研究还在感染早期（即从基线至血清HBV DNA峰值阶段）识别出共变异模式。各亚组的保守共变异模式（即代表性共变异模式）通过树形结构进行可视化呈现。本研究结果表明，识别出的共变异模式与病毒动力学、血清转换及基因型密切相关。此外，即使患者在HBV感染早期无明显临床症状，这些代表性共变异模式也可帮助临床医师为患者制定适宜的治疗方案。