Hepatitis B virus reverse transcriptase polymorphisms between treated and treatment-naïve chronically infected patients

Abstract

The aim of this study was investigation of variation(s) in the Hepatitis B virus (HBV) reverse transcriptase domain. 120 patients with chronic HBV infection recruited. 104 patients were received nucleos(t)ide analogs treatments. DNA extractions were done from plasma samples. Direct sequencing and alignment of Polymerase Chain Reaction products were applied for further analysis. HBV genotypes determined by NCBI's Genotyping Tool. Polymorphism(s) were detected by using DnaSP software. Of 120 samples, 98 were sequenced. All of products were HBV genotype D. 13/98 (13.27%) of patients had M539I/V substitutions corresponding to YMDD motif. FLLAQ to FLMAQ was observed among 22/98 (22.98) patients. Two substitutions N459Y and L515M were significantly correlated (R² = 0.486 and R² = 0.941 respectively) with FLLAQ motif variation. Mutation ratio among treatment-received patients to treatment-naïve patients was 0.2-0.6. Drug resistance conferring substitutions (DRCSs) were rtL180M (22/98), rtA194V (11/98), rtM204V (1/98), and rtM204I (11/98). Furthermore, six variants were observed among all patients. Appearance of DRCSs in HBV polymerase is a major obstacle to the virus treatments. In the present study, it was shown that DRCSs are more prevalent among treated patients. Therefore, replacement of current anti-viral regimen with novel anti-HBV drugs is warranted in the future.

Keywords: Chronic hepatitis B virus; Drug resistance conferring substitution; HBV reverse transcriptase; Hepatitis B virus polymerase; Nucleotide analogues resistance mutation.

Fig. 1

Bulk substitution among chronically HBV infected males and females treated either with LAM or ADV

Fig. 2

FI score of HBV polymerase substitution. Substitutions with low FI were Y592F (0.895), Y592H (0.895), E598N (1.15), H599R (1.67), Q602H (0.805), Q602P (1.15), K603R (1.845), R609I (1.525), L611H (1.67), P616T (1.845), C622G (1.845), I625M (1.67), V626G (1.525), L628F (1.67), G630A (1.67), F631L (1.845), and A632C (1.445). L629G/W had medium functional impact with FI score of 1.975. Further neutral substitutions with FI score of 0 were G442L, S444P, L457F, N459H/Y, M464L, P465Q/H, D466K/N, Y470S/L, L481S, H491Q, F501L, R502Q, L515M, A521V, I522L, V526I, A529V, M539V, D541E, S548T/N, V549A/E, Q550H/S/P, L552H/R, S554A, F556Y, V559A, I568V, N573H/S, K574R, K576Q, Y580H, N583H, I589G, E598D, and I601R. Further neutral substitutions were C591S (− 1.1), I601K/M (0.69), I601V (− 0.625), Q602L (0.255), E606D/K (0.345), and I613V (− 1.355)

Fig. 3

Partial correlation of LAM or ADV treatments with investigated HBV polymerase substitutions. The circles are marked with R-square values. Most notable correlated substitutions with LAM were M539I (R² = 0.306; p = 0.001), F556Y (R² = 0.185; p = 0.034), Y592H (R² = − 0.245; p = 0.007), and I601V (R² = − 0.195; p = 0.027). Further substitutions correlated with ADV were also comprised of Q602L (R² = 0.161; p = 0.056) and L629G (R² = 0.193; p = 0.028)

Fig. 4

Diagram of mutations within treated and treatment-naïve population groups. Synonymous mutations are shown in Green and non-synonymous in Red. Synonymous A747C mutations were existed in all groups. Five other non-synonymous mutations, including A677T, G728A, T752C, A762G, and T771A were also identical in the population groups (color figure online)