Occult HBV infection in Chinese blood donors: role of N-glycosylation mutations and amino acid substitutions in S protein transmembrane domains

Abstract

Occult hepatitis B virus infection (OBI) is a low-level asymptomatic phase of HBV infection. Evidence of OBI clinical relevance is emerging but the mechanisms of its occurrence remain unclear. In this study, the molecular characteristics of 97 confirmed OBI from Chinese blood donors were analyzed and relevant mutations were identified. Recombinant HBsAg bearing these mutations were expressed in vitro and the antigenicity and HBsAg secretion properties were analyzed. Results showed that 45 (46.4%) genotype B, 50 (51.5%) genotype C, and 2 (2.1%) genotype D sequences were identified. Two groups of mutations in the S gene were significantly associated with OBI. The first group included mutations creating new N-linked glycosylation sites at positions s116, s123, s130, and s131 + s133 or removing the existing one at s146. Mutations TCT123-125NCT/NFT were associated with reduced antigenicity, while TST116-118NST, GTS130-132NTS, and TSM131-133NSS/NYT/NST were associated with varying levels of impaired HBsAg secretion. N146 mutations had no effect on HBsAg production pattern. The second group included substitutions within the S transmembrane domains TMD1-3. Only mutations C85R, L87R, L88R, and C90R within TMD2 were associated with defective HBsAg production. These mutations appear to be rare and mostly strain specific but they may contribute to the multifactorial occurrence of OBI.

Occult hepatitis B infection and hepatocellular carcinoma: Epidemiology, virology, hepatocarcinogenesis and clinical significance

“…Studies have shown that some patients with OBI harbour a higher proportion of mutations in the preS/S region than patients with CHB, which may result in a reduced antigenicity for HBsAg detection or impairment in HBsAg production or secretion. 7,12,[36][37][38][39][40][41] The mutations in pre-S1 may also alter the B and T epitopes that affect immune recognition. 7 However, HBV DNA isolated from individuals with OBI is fully replication competent in vitro, 42 and the virus can also be transmitted via blood transfusion or organ transplantation.…”

N-Glycosylation and N-Glycan Processing in HBV Biology and Pathogenesis

“…Since misfolded proteins are usually retained within cells by the ER protein quality control [ 15 ], the altered antigenicity of hyper-glycosylated HBsAg may be attributed to epitope shielding rather than to mutation-induced conformational changes. With this in mind, it is not surprising that N-glycosylation mutations are increasingly reported in individuals with occult HBV infection (OBI), characterized by sustained viral replication in the absence of detectable HBsAg [ 59 , 61 , 64 ] ( Table 1 ). This observation has great clinical relevance, highlighting the importance of complementary assays for accurate diagnosis of HBV infection in HBsAg-negative patients.…”

Molecular characteristics of HBV infection among blood donors tested HBsAg reactive in a single ELISA test in southern China

“…More specifically, blood with HBsAg ELISA R-NR and NAT NR results may place recipients at risk of HBV transmission, due to the extremely low levels of HBsAg and HBV DNA, respectively. Previous studies have shown that mutations in the S region and basic core promoter (BCP) and pre-core (PC) of HBV can affect the detection of HBsAg and viral nucleic acid [ 3 , 4 ].…”