Background:  Hepatitis B virus (HBV) is an important cause of morbidity and mortality among HIV⁺ individuals. Occult HBV is defined as the presence of HBV DNA in the absence of detectable hepatitis B surface antigen (HBsAg) in the serum. Multiple distinct HBV genotypes have been described. In addition, multiple viral variants exist within an individual (viral quasispecies). Virologic characterization of occult HBV, particularly during HIV co-infection, has not been well studied. We hypothesized that unique mutations are present in HBV DNA from subjects with occult HBV infection.

Methods: Region-specific nested polymerase chain reaction (PCR) assays were developed for HBV pre-surface (PreS, 542 bp) and surface (S, 356 bp) that amplify multiple HBV genotypes at varying DNA levels. HBV infections were evaluated by real-time PCR for serum HBV levels and occult HBV infections were determined by standard serologic assays as previously described. To identify mutations unique to occult infection, the PreS and surface regions from 7 chronic infections and 6 occult infections were amplified and at least 10 clones per region per individual were sequenced. Phylogenetic analysis was performed to determine the HBV genotype and quasispecies parameters such as genetic distance, entropy, and dN/dS ratio were calculated. Genotype matched sequence analysis was performed to identify occult-associated mutations.

Results:  HBV DNA levels ranged from 113 to 2.6x10⁶ IU/mL for chronic HBV infections and from 109 to 0.76x10⁶ IU/mL for occult HBV infections. Quasispecies diversity calculations showed that multiple distinct variants were found in the PreS and S regions for all chronics and occults analyzed. Furthermore, the PreS region was generally more diverse than the S region. Dual infection with genotypes A and G PreS variants was also identified in one occult HBV infection. Sequence analysis identified novel mutations in subjects with occult HBV infection—for genotype A, 2 for genotype E, and 6 for genotype G—as well as several previously published mutations in the antigenic determinant of HBsAg. Several novel occult mutations were also identified in the PreS region—6 for genotype A and 13 for genotype G.

Conclusions: Unique mutations in HBV DNA PreS and S regions from subjects with occult HBV infection were identified. We speculate that mutations in these regions may play a role in antigenic binding and detection with serologic assays.