Amino acid similarities and divergences in the small surface proteins of genotype C hepatitis B viruses between nucleos(t)ide analogue-naïve and lamivudine-treated patients with chronic hepatitis B

• SHBs sequences from LMV-treated C-genotype HBV harbor significant higher mutation frequencies.
• LMV increases mutations in MHR (except ‘a’ determinant) and TM 3/4 of SHBs.
• LMV induces antigenicity-altering SHBs/RT mirror changes outside ‘a’ determinant.
• Frequency of sW182stop increases and sC69stop decreases after LMV therapy.
• Key sites in conserved SHBs regions identified here remain intact after LMV therapy.

Entire C-genotype small hepatitis B surface (SHBs) sequences were isolated from 139 nucleos(t)ide analogues (NA)-naïve and 74 lamivudine (LMV)-treated chronic hepatitis B (CHB) patients. The conservation and variability of total 226 amino acids (AAs) within the sequences were determined individually, revealing significant higher mutant isolate rate and mutation frequency in LMV-treated cohort than those in the NA-naïve one (P = 0.009 and 0.0001, respectively). Three absolutely conserved fragments (s16–s19, s176–s181 and s185–s188) and seven moderately conserved regions (a few AA sites acquiring increased variability after LMV-treatment) were identified. The significant mutation rate increase after LMV-treatment occurred primarily in major hydrophilic region (except ‘a’ determinant) and transmembrane domain 3/4, but not in other upstream functional regions of SHBs. With little influence on immune escape-associated mutation frequencies within ‘a’ determinant, LMV-monotherapy significantly induced classical LMVr-associated mirror changes sE164D/rtV173L, sI195M/rtM204V and sW196L/S/rtM204I, as well as non-classical ones sG44E/rtS53N, sT47K/A/rtH55R/Q and sW182stop/rtV191I outside ‘a’ determinant. Interestingly, another newly-identified truncation mutation sC69stop/rtS78T decreased from 7.91% (11/139) in NA-naïve cohort to 2.70% (2/74) in LMV-treated one. Altogether, the altered AA conservation and diversity in SHBs sequences after LMV-treatment in genotype-C HBV infection might shed new insights into how LMV-therapy affects the SHBs variant evolution and its antigenicity.