Huang Fung-Yu, Wong Danny Ka-Ho, Seto Wai-Kay, Zhang An-Ye, Lee Cheuk-Kwong, Lin Che-Kit, Fung James, Lai Ching-Lung, Yuen Man-Fung
Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China.
Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China; State Key Laboratory for Liver Research, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China.
PLoS One. 2014 Jun 5;9(6):e99028. doi: 10.1371/journal.pone.0099028. eCollection 2014.
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.
由于定义该疾病状态的DNA水平极低，乙肝表面抗原（HBsAg）阴性的乙型肝炎病毒（HBV）感染的潜在机制极难阐明。我们采用了一种高效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。
对185份来自HBsAg阴性、HBV DNA阳性受试者或已知有HBsAg血清学清除的慢性乙型肝炎（CHB）受试者的血清以及60份肝活检组织进行滚环扩增，随后进行全长HBV基因组测序。纳入11名HBsAg阳性的CHB受试者作为对照。分析了在调控区域鉴定出的关键突变对启动子活性的影响。
分别从HBsAg阴性受试者和对照受试者中扩增出22个和11个全长HBV基因组。HBV基因型C是优势毒株。无论基因型如何，HBsAg阴性受试者中的突变频率均高于对照。与对照相比，HBsAg阴性受试者整个HBV基因组的核苷酸多样性显著更高（p = 0.008），与49份CHB患者的参考序列相比也更高（p = 0.025）。此外，HBsAg阴性受试者在四个病毒基因中的氨基酸替换显著多于对照（所有p<0.001）。许多突变仅在HBsAg阴性受试者中发现，包括启动子区域的缺失（13.6%）、前S2/S起始密码子的缺失（18.2%）、前S2/S mRNA剪接位点的破坏（4.5%）、核苷酸重复（9.1%）以及“α”决定簇区域的错义突变，这些都导致了HBsAg产生的缺陷。
这些数据表明，多种限制病毒转录活性的突变积累导致了HBV感染中HBsAg阴性。
