bOccult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surface antigen (HBsAg) outside the preseroconversion window period. The mechanisms leading to OBI remain largely unknown. The potential role of specific amino acid substitutions in the S protein from OBI in HBsAg production and excretion was examined in vitro. HBsAg was quantified in culture supernatants and cell extracts of HuH-7 cells transiently transfected with plasmids containing the S gene of eight HBsAg ؉ controls and 18 OBI clones. The intracellular (IC)/extracellular (EC) HBsAg production ratio was ϳ1.0 for the majority of controls. Three IC/EC HBsAg patterns were observed in OBI strains clones: pattern 1, an IC/EC ratio of 1.0, was found in 5/18 OBI clones, pattern 2, detectable IC but low or undetectable EC HBsAg (IC/EC, 7.0 to 800), was found in 6/18 OBIs, and pattern 3, low or undetectable IC and EC HBsAg, was found in 7/18 clones. Intracellular immunofluorescence staining showed that in pattern 2, HBsAg was concentrated around the nucleus, suggesting retention in the endoplasmic reticulum. The substitution M75T, Y100S, or P178R was present in 4/6 pattern 2 OBI clones. Site-directed-mutagenesis-corrected mutations reversed HBsAg excretion to pattern 1 and, when introduced into a control clone, induced pattern 2 except for Y100S. In a control and several OBIs, variants of a given quasispecies expressed HBsAg according to different patterns. However, the P178R substitution present in all cloned sequences of two OBI strains may contribute significantly to the OBI phenotype.
O ccult hepatitis B virus (HBV) infection/carriage (OBI) ischaracterized by the presence of very low levels of HBV DNA in plasma and/or in liver with hepatitis B surface antigen (HBsAg) being undetectable using the most sensitive commercial assays, with or without antibodies to hepatitis core antigen (anti-HBc) or hepatitis B surface antigen (anti-HBs), outside the preseroconversion window period (1). OBI represents a particular form of persistent or chronic infection that is encountered globally, albeit at different frequencies depending on endemicity and genotype (2). OBI is a potential source of HBV transmission by transfusion and organ transplantation, can reactivate in association with immunodeficiency or immunosuppressive treatments, and might be a risk factor for liver cancer (2). Several mechanisms have been proposed as responsible for OBI, such as imperfect control by the host immune system (3, 4), multiple amino acid substitutions in the S protein affecting HBsAg detection with commercial immunoassays (5, 6), mutations in regulatory elements negatively affecting virus replication (7, 8), and mutations affecting posttranscriptional mechanisms regulating S protein expression (9, 10).A high frequency of mutations has been observed especially within the major hydrophilic region (MHR) of S protein from OBI strains, and these may alter antigenicity, HBV infectivity, cell tropism, and virion morphogenesis (4-6, 11). In...