Naturally occurring nucleos(t)ide analogue resistance (NUCr) substitution frequencies in the reverse transcriptase (RT) of the hepatitis B virus (HBV) were studied extensively after the clinical approval of nucleos(t)ide analogues (NUCs; year of approval 1998). We aimed to study NUCr substitutions in HBV RT sequences obtained before 1998 and better understand the evolution of RT sequences without NUC pressures. Our strategy was to retrieve HBV sequences from GenBank deposited before 1998. The initial search used the keywords "hepatitis B virus" or "HBV" and 1139 sequences were found. Data analyses included information extraction: sequence quality control and amino acid substitution analysis on 8 primary NUCr and 3 secondary substitution codons. Three hundred and ninety-four RT-containing sequences of 8 genotypes from 25 countries in 4 continents were selected. Twenty-seven (6.9%) sequences were found to harbor substitutions at NUCr-related codons. Secondary substitutions (rtL80V and rtV173G/A/L) occurred more frequently than primary NUCr substitutions (rtI169L; rtA181G; T184A/S; rtS202T/R; rtM204L and rtM250K). Typical amino acid substitutions associated with NUCr were of rtL80V, rtV173L and rtT184A/S. We confirm the presence of naturally occurring typical HBV NUCr substitutions with very low frequencies, and secondary substitutions are more likely to occur than primary NUCr substitutions without the selective pressure of NUCs. Keywords: hepatitis B virus; naturally occurring; nucleos(t)ides analogue resistance; pre-existing; reverse transcriptase; substitution.

Background & aims: As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels. Methods: Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed. Results: Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg. Conclusion: The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements.