title: Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  These data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.
authors.affiliation: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China.
authors.forename: Fung-Yu
authors.initials: FY
authors.lastname: Huang
authors.affiliation: 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.
authors.forename: Danny Ka-Ho
authors.initials: DK
authors.lastname: Wong
authors.affiliation: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China.
authors.forename: Wai-Kay
authors.initials: WK
authors.lastname: Seto
authors.affiliation: Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, China.
authors.forename: An-Ye
authors.initials: AY
authors.lastname: Zhang
authors.affiliation: Hong Kong Red Cross Blood Transfusion Service, Hospital Authority, Hong Kong, China.
authors.forename: Cheuk-Kwong
authors.initials: CK
authors.lastname: Lee
authors.affiliation: Hong Kong Red Cross Blood Transfusion Service, Hospital Authority, Hong Kong, China.
authors.forename: Che-Kit
authors.initials: CK
authors.lastname: Lin
authors.affiliation: 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.
authors.forename: James
authors.initials: J
authors.lastname: Fung
authors.affiliation: 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.
authors.forename: Ching-Lung
authors.initials: CL
authors.lastname: Lai
authors.affiliation: 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.
authors.forename: Man-Fung
authors.initials: MF
authors.lastname: Yuen
mesh_terms: D000328:Adult; D000368:Aged; D002681:China; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D005260:Female; D014644:Genetic Variation; D016679:Genome, Viral; D005838:Genotype; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D019694:Hepatitis B, Chronic / Q000175:diagnosis / Q000821:virology*; D006801:Humans; D008099:Liver / Q000473:pathology / Q000821:virology; D008297:Male; D008875:Middle Aged; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000737:chemistry; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D012367:RNA, Viral