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Occult hepatitis B virus (HBV) infection (OBI) has non-negligible clinical significance, but the mechanism of its occurrence remains unclear. Growing evidence suggests that mutations in the pre-S region of HBV genome may be associated with the occurrence of OBI. However, the role of pre-S mutations in OBI and its molecular mechanism was not fully understand. Here, the pre-S sequences from 307 OBI blood donors and 293 hepatitis B surface protein (HBsAg)-positive blood donors were obtained, and we observed a higher frequency of naturally occurring pre-S mutations in OBI donors infected with genotype B/C HBV than in HBsAg-positive donors, suggesting their potential positive role in OBI. In both genotype B and C, several pre-S mutants resulted in markedly reduced HBsAg production in vitro. In particular, the T68I, S78N and N98T mutants of genotype B were proven to significantly decrease the HBsAg synthesis by affecting the pre-S2/S promoter activity, and thereby promoting the occurrence of OBI.

Background The high prevalence of hepatitis C virus (HCV) infection and the resulting burden of the disease are significant issues to public health worldwide. Although direct-acting antiviral drugs (DAAs) with good tolerance and bioavailability are available, resistance-associated substitutions (RASs) often jeopardize the successful sustainment of virological responses in HCV treatment. High-frequency baseline RASs in treatment-naive patients can lead to failures in DAA treatment. Clinical data on HCV RASs in patients from China are limited and require investigations. Methods 262 HCV RNA positive plasma from Chinese blood donors were genotyped and amplified with subtype-specific primers for NS3 and NS5A regions. RASs were analyzed using Geno2pheno. The codon usage of each resistance-associated substitution was calculated for genetic barrier analysis. Results The two main subtypes in mainland China were 1b and 2a, followed by subtype 6a, 3b, 3a, and 1a. In NS3 region of 1b subtype, substitutions (T54S, V55A, Y56F, Q80 K/L, S122 G/T, R117 H/C, V170I and S174A) were present in 89.7% (96/107) of the samples. Other RASs (M28L, R30Q, P58 L/S and Y93H) were observed in 22.1% (25/113) of the samples in NS5A region. A crucial RAS, Q80K, and two other mutations (S122G + V170I) was identified in the same sequence, which reduced its susceptibility to protease inhibitor ASV and resulted in resistance to SMV. In NS5A, Y93H was detected in 9.7% (11/113) of the 1b samples, leading to medium-to-high level resistance to all six commercialized NS5A inhibitors. S122G-NS3 and Y93H-NS5A occurred simultaneously in 38.1% (7/22) of the samples with mutations in both two regions. Moreover, codon usage of S122G-NS3 and Y93H-NS5A revealed that both variants had the lowest genetic barrier and required only one transition to confer resistance. Conclusions Low genetic barriers facilitated the generation of resistance mutants and threated the efficacy of DAA regimens. The baseline RASs posed a great challenge to real-world DAA application.