The dynamic changes of HBV quasispecies diversity in infancy after immunoprophylaxis failure: a prospective cohort study

Abstract

Background: Previous works have observed that younger infants with chronic hepatitis B virus (HBV) infection are more responsive to antiviral treatment. However, the underlying mechanism remains unclear. In this study, the dynamic changes of HBV quasispecies in infants with immunoprophylaxis failure were investigated to provide virological explanations for clinical management on infantile antiviral therapy.

Methods: Thirteen 7-month-old infants with immunoprophylaxis failure and their mothers were enrolled from a prospective cohort, and 8 of them were followed up to 3 years old. The sequences of HBV quasispecies were analyzed by the full-length genome clone-based sequencing, and compared among mothers and their infants at different ages.

Results: The results revealed that the complexity, mutation frequency and genetic distance of HBV quasispecies decreased significantly at full-length, partial open reading frames and regulatory regions of HBV genome at nucleotide level in 7-month-old infants comparing with their mothers, whereas increased significantly to near the maternal level when infants grew up to 3 years old. Furthermore, similar changes were also found in Core, PreS2, RT and P regions of HBV genome at amino acid level, especially for potential NAs-resistant mutants in RT region and immune-escape mutants in Core and PreS2 regions.

Conclusions: This study uncovered the evolution of HBV quasispecies in infancy after mother-to-child transmission, which may provide the virological evidence for explaning that younger children are more responsive to antiviral therapy.

Keywords: Hepatitis B virus; Immunoprophylaxis failure; Infantile antiviral therapy; Mother-to-child transmission; Quasispecies.

Fig. 1

Comparative analysis of HBV quasispecies characteristics during MTCT. The quasispecies complexity, mutation frequency and genetic distance were analyzed between mothers and their paired 7-month-old infants at nucleotide level (A, B and C) and amino acid level (D, E and F), respectively. The comparison of synonymous substitution rate (dS) (G) and non-synonymous substitution rate (dN) (H). *represents P < 0.05. **represents P < 0.01. “nt” represents the nucleotide level. “aa” represents the amino acid level. NTCP-BD: sodium taurocholate cotransporting polypetide binding domain. MHR: major hydrophilic region. Quasispecies complexity was measured using normalized Shannon entropy (Sn). Genetic distance was calculated at the nucleotide level under Tamura 3-parameter method and at the amino acid level under Jones–Taylor–Thornton matrix-based method. dS and dN were calculated under modified Nei–Gojobori model with Jukes–Cantor correction

Fig. 2

Comparative analysis of serum markers and HBV quasispecies characteristics between 8 pairs of mothers and infants at 7 months and 3 years old. Dynamic change of serum markers and HBV quasispecies characteristics at full-length HBV genome level (A). The quasispecies complexities (B), mutation frequencies (C) and genetic distances (D) of Core, PreS2, P, RT, S and NTCP-BD regions at nucleotide level at three time points. The quasispecies complexities (E), mutation frequencies (F) and genetic distances (G) of Core, PreS2, P and RT regions at amino acid level at three time points. The dynamic change of synonymous substitution rates (dS) (H) and the non-synonymous substitution rates (dN) (I) of Core, PreS2, P, RT, S and NTCP-BD regions at three time points. The phylogenetic trees of 8 mothers and their paired infants at 7 months and 3 years old. (J) Black point represents the clones from mothers, light blue point for 7-month-old infants and dark blue point for 3-year-old infants. *represents P < 0.05. “nt” represents nucleotide level. “aa” represents amino acid level. NTCP-BD: sodium taurocholate cotransporting polypetide binding domain; Quasispecies complexity was measured using normalized Shannon entropy (Sn). Genetic distance was calculated at the nucleotide level under Tamura 3-parameter method and at the amino acid level under Jones–Taylor–Thornton matrix-based method. dS and dN were calculated under modified Nei–Gojobori model with Jukes–Cantor correction

Fig. 3

Comparative analysis of the potential NAs-resistant mutations in RT region and the mutation rates of single amino acid site in Core and PreS2 regions. Dynamic changes of the indel mutation rates in Core and RT regions of HBV genome from mothers and infants at 7 months and 3 years old. (A) The cumulative rate of NAs-resistant mutations in RT region of HBV genome from mothers and infants at 7 months and 3 years old. (B) The ratio of clones with NAs-resistant mutations in RT region of HBV genome from mothers and infants at 7 months and 3 years old. (C) The single amino acid site mutation rate in Core and PreS2 region of all clones from mothers and infants at 7 months and 3 years old. (D) Black line represents the data from mothers, light blue line for 7-month-old infants and dark blue line for 3-year-old infants. Sites with mutation rate > 1% in Core region and sites with mutation rate > 0.5% in PreS2 region were noted. All the mutations were defined based on a same consensus sequence synthesized by all clones from mothers

Fig. 4

Graphic abstract for the dynamics of hepatitis B virus quasispecies after MTCT and evolution in infancy