Abstract
BackgroundThe 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.MethodsA 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.Results22 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.ConclusionsThese data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.
Highlights
Hepatitis B virus (HBV) infection leads to a wide spectrum of liver injuries, ranging from acute self-limiting infection and fulminant hepatitis to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) [1]
Spontaneous loss of HBV surface antigen (HBsAg) is a rare event in chronic hepatitis B (CHB) infection [3,4,5,6]
The continued presence of detectable HBsAg in serum is a prominent feature of HBsAg-positive CHB infection or overt CHB infection, HBsAg seroclearance may not signify eradication of hepatitis B virus (HBV)
Summary
Hepatitis B virus (HBV) infection leads to a wide spectrum of liver injuries, ranging from acute self-limiting infection and fulminant hepatitis to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) [1]. The introduction of sensitive HBV DNA detection tests has revealed the existence of HBsAg-negative HBV infection. This new entity of HBV infection is defined as ‘‘the presence of viral DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for the HBsAg’’ [9,10]. Some of these subjects may be people with or without prior medical history of HBV infection can develop to the occult phase [11]. 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
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