Occult infection related hepatitis B surface antigen variants showing lowered secretion capacity

Abstract

Aim: To elucidate the molecular mechanisms underlying hepatitis B virus (HBV) occult infection of genotype C.

Methods: A total of 10 types of hepatitis B surface antigen (HBsAg) variants from a Korean occult cohort were used. After a complete HBV genome plasmid mutated such that it does not express HBsAg and plasmid encoding, each HBsAg variant was transiently co-transfected into HuH-7 cells. The secretion capacity and intracellular expression of the HBV virions and HBsAgs in their respective variants were analyzed using real-time quantitative polymerase chain reaction assays and commercial HBsAg enzyme-linked immunosorbent assays, respectively.

Results: All variants exhibited lower levels of HBsAg secretion into the medium compared with the wild type. In particular, in eight of the ten variants, very low levels of HBsAg secretion that were similar to the negative control were detected. In contrast, most variants (9/10) exhibited normal virion secretion capacities comparable with, or even higher than, the wild type. This provided new insight into the intrinsic nature of occult HBV infection, which leads to HBsAg sero-negativeness but has horizontal infectivity. Furthermore, most variants generated higher reactive oxidative species production than the wild type. This finding provides potential links between occult HBV infection and liver disease progression.

Conclusion: The presently obtained data indicate that deficiency in the secretion capacity of HBsAg variants may have a pivotal function in the occult infections of HBV genotype C.

Keywords: Genotype C; Hepatitis B surface antigen; Hepatitis B virus; Occult infection; Reactive oxidative species; Variants.

Figure 1

Schematic experimental strategy used in this study. Briefly, one microgram of pHY92-1.1x-HBV-small S (-) having a full-genome of hepatitis B virus (HBV) with a knock-out small surface open reading frame (ORF) was co-transfected with three micrograms of pIRES2-EGFP-HBV-small S expressing the sub-genome of small surface region, which has ten variants including mock and wild-type into HuH-7 cell line transiently. After co-transfection, HuH-7 cells were incubated for 2 d. Supernatant and lysed pellet were collected and used for various assays. The tests were performed in triplicate. HBsAg: Hepatitis B surface antigen; ELISA: Enzyme linked immunosorbent assay; Q-PCR: Quantitative polymerase chain reaction; ROS: Reactive oxygen species; Neo/Kan R: Neomycin and Kanamycin resistance.

Figure 2

Secretion capacity and viral DNA formation of occult hepatitis B surface antigen variants. Extracellular secreted hepatitis B surface antigen (HBsAg) and intracellular expressed HBsAg from cell lysate were measured using a commercial HBsAg enzyme linked immunosorbent assay kit normalized via a β-galactosidase assay. After purification of the viral DNA from the supernatant and cell lysate using a total viral DNA preparation kit, detection of the viral DNA from both intracellular and extracellular was performed using real-time quantitative DNA-polymerase chain reaction assays. The hepatitis B virus DNA was normalized via a β-galactosidase assay. The tests were performed in triplicate (mean ± SD). sAg: Surface antigen. ^aP < 0.05, ^bP < 0.01 vs wild type (Nor) group.

Figure 3

Effect of occult hepatitis B surface antigen variants on the reactive oxygen species system. The reactive oxygen species level according to the occult hepatitis B virus variants in transient transfected HuH-7 was measured using 20 μmol/L of DHR123 reagent, which can detect reactive oxygen species (ROS) such as peroxide and peroxynitrite. The tests were performed in triplicate (mean ± SD). ^aP < 0.05, ^bP < 0.01 vs wild type (Nor) group.