Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection.
Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection.
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DOI：
10.1371/journal.pone.0099028
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Yuen MF
中科院分区：
文献类型：
--
作者：
Huang FY;Wong DK;Seto WK;Zhang AY;Lee CK;Lin CK;Fung J;Lai CL;Yuen MF
众所周知，HBsAg 阴性乙型肝炎病毒 (HBV) 感染的根本机制很难阐明，因为定义该病症的 DNA 水平极低。我们使用高效的扩增方法来克服这一障碍，并实现了我们的目标，即识别与该实体相关的特定突变或序列变异。通过滚环扩增和全长 HBV 基因组测序，对来自 HBsAg 阴性、HBV DNA 阳性受试者或 HBsAg 血清清除的已知慢性乙型肝炎 (CHB) 受试者的总共 185 份血清和 60 份肝活检进行了扩增。十一名 HBsAg 阳性 CHB 受试者被纳入对照。分析了调节区上鉴定的关键突变对启动子活性的影响。分别从 HBsAg 阴性和对照受试者中扩增出 22 个和 11 个全长 HBV 基因组。乙肝病毒C基因型为优势株。无论基因型如何，HBsAg 阴性受试者的突变频率均高于对照组。与对照组相比 (p = 0.008) 以及与来自 CHB 患者的 49 个参考序列 (p = 0.025) 相比，HBsAg 阴性受试者的整个 HBV 基因组的核苷酸多样性显着更高。此外，HBsAg 阴性受试者的四种病毒基因中的氨基酸取代显着高于对照组（所有 p<0.001）。许多突变是在 HBsAg 阴性受试者中独特发现的，包括启动子区域的缺失 (13.6%)、前 S2/S 起始密码子的废除 (18.2%)、前 S2/S mRNA 剪接位点的破坏 (4.5%)、核苷酸重复 (9.1%) 和“α”决定簇区域的错义突变，导致 HBsAg 产生缺陷。这些数据表明，限制病毒转录活性的多种突变的积累导致 HBV 感染中 HBsAg 阴性。
The underlying mechanism of HBsAg-negative hepatitis B virus (HBV) infection is notoriously difficult to elucidate because of the extremely low DNA levels which define the condition. We used a highly efficient amplification method to overcome this obstacle and achieved our aim which was to identify specific mutations or sequence variations associated with this entity. A total of 185 sera and 60 liver biopsies from HBsAg-negative, HBV DNA-positive subjects or known chronic hepatitis B (CHB) subjects with HBsAg seroclearance were amplified by rolling circle amplification followed by full-length HBV genome sequencing. Eleven HBsAg-positive CHB subjects were included as controls. The effects of pivotal mutations identified on regulatory regions on promoter activities were analyzed. 22 and 11 full-length HBV genomes were amplified from HBsAg-negative and control subjects respectively. HBV genotype C was the dominant strain. A higher mutation frequency was observed in HBsAg-negative subjects than controls, irrespective of genotype. The nucleotide diversity over the entire HBV genome was significantly higher in HBsAg-negative subjects compared with controls (p = 0.008) and compared with 49 reference sequences from CHB patients (p = 0.025). In addition, HBsAg-negative subjects had significantly higher amino acid substitutions in the four viral genes than controls (all p<0.001). Many mutations were uniquely found in HBsAg-negative subjects, including deletions in promoter regions (13.6%), abolishment of pre-S2/S start codon (18.2%), disruption of pre-S2/S mRNA splicing site (4.5%), nucleotide duplications (9.1%), and missense mutations in “α” determinant region, contributing to defects in HBsAg production. These data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.
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