Phylogenetic Analysis and Emerging Drug Resistance against Different Nucleoside Analogues in Hepatitis B Virus Positive Patients

Abstract

Several nucleotide analogues have been approved for use in treating hepatitis B virus (HBV) infection. Long-term exposure to therapy leads to the emergence of mutations within the HBV DNA polymerase gene, resulting in drug resistance, a major factor contributing to therapy failure. Chronic HBV patients from the Khyber Pakhtunkhwa province, Pakistan, who had completed 6 months of therapy participated in this study. Samples were collected from 60 patients. In this study, the entire reverse transcriptase domain of the HBV polymerase gene was amplified using nested polymerase chain reaction and sequenced. Drug-resistant mutations were detected in nine (22.5%) patients. All of these patients had lamivudine-resistant mutations (rtM204V + L180M), while seven individuals (17.5%) had both lamivudine- plus entecavir-resistant mutations (L180M + M204V + S202G). N236T, a mutation that gives rise to tenofovir and adefovir resistance, was observed in two (5%) patients. T184A, a partial drug-resistant mutation to entecavir, was found in five (12.5%) patients. Furthermore, other genotypic variants (100%) and vaccine escape mutations (5%) were additionally observed. Moreover, pN459Y (35%), pN131D (20%), pL231S (20%), pP130Q (17.5%), pS189Q (12.5%), pP161S (5%), pH160P (2.5%), pT322S (2.5%), and pA223S (2.5%) mutations in the polymerase gene, as well as sA166V (17.5%), sQ181K (12.5%), sV184R (7.5%), sA17E (5%), sP153S/K (5%), sW156C (5%), sC76Y (2.5%), and S132F (2.5%) mutations in the small surface gene, were identified for the first time in this study. Phylogenetic analysis showed that genotype D was predominant amongst the HBV carriers. Subtype D1 was found in most patients, while two patients were subtype D9. These novel findings may contribute to the body of knowledge and have clinical significance for treating and curing HBV infections in Pakistan.

Keywords: genotypes; hepatitis B virus; mutations; phylogenetic analysis; polymerase gene; surface gene.

Figure 1

Phylogenetic Tree Representing Clustering of Study Samples with Genotype D. A maximum likelihood method and Tamura-Nei model were used to generate a phylogenetic tree representing the HBV sequences found in 40 HBV-positive patients. The bootstrap value was shown with blue dots. Nodes with 70% confidence were included and the lower were removed. The HBV sequences were aligned with different HBV genotype reference sequences using ClustalW. This analysis involved 62 nucleotide sequences. There was a total of 5131 positions in the final dataset. Evolutionary analyses were conducted using MEGA X. Light pink color represent patient samples. The genotypes are as follows: Genotype A (light yellow), Genotype B (light purple), Genotype C (light orange), Genotype D (Cyan), Genotype E (light green), Genotype F (Green), Genotype G (green) and Genotype H (blue). Each sequence’s accession numbers and genotype were mentioned in the phylogenetic tree. The clustering indicates that genotype D is the most common genotype in our study cohort.

Figure 2

Phylogenetic tree identifying the HBV sub-genotypes in the study cohort. Evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. This analysis involved 81 nucleotide sequences. The bootstrap value was shown with blue dots. Nodes with 70% confidence were included and the lower were removed. Patient samples are represented by light yellow color. Each sequence’s accession numbers, sub-genotype and country of origin were mentioned in the phylogenetic tree. The clustering indicates that sub-genotype D1 is the most common genotype in our study cohort. Two patient samples clustering with sub-genotype D9 showed the appearance of novel subtype in Pakistan. The evolutionary analyses were conducted in MEGA X.