Discovery of a Novel Mutation (X8Del) Resulting in an 8-bp Deletion in the Hepatitis B Virus X Gene Associated with Occult Infection in Korean Vaccinated Individuals.

Universal infantile hepatitis B virus (HBV) vaccination may lead to an increase in vaccine escape variants, which may pose a threat to the long-term success of massive vaccination. To determine the prevalence of occult infections in Korean vaccinated individuals, 87 vaccinated subjects were screened for the presence of HBV DNA using both the nested PCR protocol and the VERSANT HBV DNA 3.0 assay. The mutation patterns of variants were analyzed in full-length HBV genome sequences. Their HBsAg secretion and replication capacities were investigated using both in vitro transient transfection and in vivo hydrodynamic injection. The presence of HBV DNA was confirmed in 6 subjects (6.9%). All six variants had a common mutation type (X8Del) composed of an 8-bp deletion in the C-terminal region of the HBV X gene (HBxAg). Our in vitro and in vivo analyses using the full-length HBV genome indicated that the X8Del HBxAg variant reduced the secretion of HBsAg and HBV virions compared to the wild type. In conclusion, our data suggest that a novel mutation (X8Del) may contribute to occult HBV infection in Korean vaccinated individuals via a reduced secretion of HBsAg and virions, possibly by compromising HBxAg's transacting capacity

Clinical and virological data from 6 vaccinated subjects demonstrated to be HBV DNA positive by both the nested PCR protocol and VERSANT HBV DNA 3.0 assay.

<p>^aThe normal range of ALT (IU/L) is less than or equal to 41.</p><p>^bAnti-HBs antibody (IU/L): non-reactive < 10 and reactive ≥ 10.</p><p>^cAnti-HBe antibody (COI): non-reactive > 1.0 and reactive ≤ 1.0.</p><p>^dThe detection limit of HBV bDNA probe (copies/ml) is greater than or equal to 2000.</p><p>Clinical and virological data from 6 vaccinated subjects demonstrated to be HBV DNA positive by both the nested PCR protocol and VERSANT HBV DNA 3.0 assay.</p

Comparison of the HBsAg secretion and virion formation capacities between the HBV full-length genome constructs pHBV1.2X-Wild and pHBV1.2X-X8Del in HuH-7.5 cells.

<p>After the transient transfection of pHBV-1.2x-Wild type and pHBV1.2X-X8Del into HuH-7.5 cells for 2 days, the levels of HBsAg (A) and HBeAg (C) from the supernatant and the RLB-lysed pellet were measured using the HBsAg and HBeAg ELISA kits. The virion levels in the supernatant and pellet after the transfection of both plasmids were compared using real-time quantitative PCR (B) and Southern blotting analysis (D). The relative intensity of DNA from the Southern blotting was measured using ImageJ software (NIH, USA) (E).</p

The location of a novel mutation (XDel8) that resulted in the deletion of eight nucleotides (nt 396 to 404) in the C-terminus of the X region found in the 6 vaccinated subjects proven to be HBV DNA positive.

<p>The location of a novel mutation (XDel8) that resulted in the deletion of eight nucleotides (nt 396 to 404) in the C-terminus of the X region found in the 6 vaccinated subjects proven to be HBV DNA positive.</p

Mutation patterns within the HBV full-length genome in the 6 vaccinated subjects verified to be HBV DNA-positive.

<p>Mutation patterns within the HBV full-length genome in the 6 vaccinated subjects verified to be HBV DNA-positive.</p

Comparison of HBsAg secretion (A) and virion formation (B) capacities between the HBV full-length genome constructs pHBV1.2X-Wild and pHBV1.2X-X8Del in the hydrodynamic mouse model.

<p>Circulating HBsAg from mouse serum was measured using the HBsAg ELISA kit. HBV DNA from virions was measured by real-time quantitative PCR.</p

Strategy of the current study for the elucidation of molecular epidemiologic features and the mechanism underlying a novel X8Del variant that lead to occult infections in Korean vaccinated individuals.

<p>Strategy of the current study for the elucidation of molecular epidemiologic features and the mechanism underlying a novel X8Del variant that lead to occult infections in Korean vaccinated individuals.</p

Phylogenetic analysis based on the HBV full-length genome sequences of eight references and the 6 Korean vaccinated subjects proven to be HBV DNA positive.

<p>The evolutionary history was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Phylogenetic analyses were conducted in MEGA4.</p