Molecular analysis of HBV pre-core gene mutations in patients co-infected with HIV at a tertiary care hospital in North India

Abstract

Objective. Hepatitis B virus (HBV) spontaneous mutations may impact the severity of liver disease. This study aimed to assess the mutations in the pre-core (PC) region in HBV-HIV (human immunodeficiency virus) co-infected patients. Additionally, we explored its association with genotypes and examined the clinical implications. Methods. A total of 100 HBV-HIV co-infected patients and 50 HBV mono-infected patients were included in the study. We focused on the PC region of the HBV genome, sequencing it to identify PC mutant variants. PCR products were quantified via spectrophotometry and sequenced using the Sanger method. The resulting sequences were assembled, annotated and aligned in a single reading frame. Subsequent mutational and phylogenetic analyses were performed using UGENE software to determine the genotypes of the isolates. Results. The PC region was successfully amplified and sequenced in 27 samples, comprising 16 from HBV-HIV co-infected patients and 11 from HBV mono-infected patients. Phylogenetic analysis identified two HBV genotypes: genotype D, which was predominant and found in 24 samples (88.9%), and genotype A, present in 3 samples (11.1%). A T-to-C mutation at nucleotide position 1912 was detected in 48.1% of the patients. Furthermore, several additional PC mutations were observed, including A1850T, C1858T, G1899A, G1862T, G1951T, T1812C and T1809G, along with novel mutations such as C1936T, A2011G, T2020A and C2044T. Notably, the prevalence of these PC mutations did not significantly differ between the HBV mono-infected and HBV-HIV co-infected groups. Conclusion. This study underscored the prevalence of PC mutations in HBV-HIV co-infected patients. Although several of these mutations have been previously reported, our findings also revealed novel variants. Further research is needed to elucidate the clinical significance of these new mutations.

Keywords: hepatitis B virus; hepatitis B virus and human immunodeficiency virus (HBV-HIV) co-infection; pre-core (PC) mutation.

Fig. 1.. Flow chart showing selection and processing of HIV/HBV co-infected and HBV mono-infected patient samples for HBV PC region mutation analysis.

Fig. 2.. Heatmap showing the presence of mutations across different genotypes.

Fig. 3.. This phylogenetic tree represents the evolutionary relationships among HBV isolates from different geographic locations and genotypes. The tree was constructed using iTOL based on nt sequence alignments. Highlight: represents the reference sequence (NCBI RefSeq) used for comparison. Genotype A is highlighted in green, and all other highlighted colours represent genotype D samples from different geographical regions around the world. The bootstrap values (black circles at nodes) indicate the reliability of branching patterns.

Fig. 4.. Summary of observed mutations in the PC region based on sequence alignments (n=27). This figure illustrates the pattern and frequency of key nt substitutions, including G1896A and A1762T/G1764A, across the patient cohort.