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. 2024 May 17;25(10):5481.
doi: 10.3390/ijms25105481.

Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation

Eva Dopico  1   2 , Marta Vila  3 , David Tabernero  3   4   5 , Josep Gregori  5 , Ariadna Rando-Segura  3   4   6 , Beatriz Pacín-Ruíz  3   4 , Laura Guerrero  1 , Itziar Ubillos  1 , Miguel J Martínez  7   8   9 , Josep Costa  7 , Josep Quer  4   5   10 , Javier Pérez-Garreta  3 , Alejandra González-Sánchez  6   9   11 , Andrés Antón  6   9   11 , Tomás Pumarola  6   9 , Mar Riveiro-Barciela  4   12 , Roser Ferrer-Costa  13   14 , Maria Buti  4   12 , Francisco Rodríguez-Frías  4   13   15 , Maria Francesca Cortese  3   4
Affiliations

Genotyping Hepatitis B virus by Next-Generation Sequencing: Detection of Mixed Infections and Analysis of Sequence Conservation

Eva Dopico et al. Int J Mol Sci. .

Abstract

Our aim was to develop an accurate, highly sensitive method for HBV genotype determination and detection of genotype mixtures. We examined the preS and 5' end of the HBV X gene (5X) regions of the HBV genome using next-generation sequencing (NGS). The 1852 haplotypes obtained were subjected to genotyping via the Distance-Based discrimination method (DB Rule) using two sets of 95 reference sequences of genotypes A-H. In clinical samples from 125 patients, the main genotypes were A, D, F and H in Caucasian, B and C in Asian and A and E in Sub-Saharan patients. Genotype mixtures were identified in 28 (22.40%) cases, and potential intergenotypic recombination was observed in 29 (23.20%) cases. Furthermore, we evaluated sequence conservation among haplotypes classified into genotypes A, C, D, and E by computing the information content. The preS haplotypes exhibited limited shared conserved regions, whereas the 5X haplotypes revealed two groups of conserved regions across the genotypes assessed. In conclusion, we developed an NGS-based HBV genotyping method utilizing the DB Rule for genotype classification. We identified two regions conserved across different genotypes at 5X, offering promising targets for RNA interference-based antiviral therapies.

Keywords: Distance-Based discrimination method; RNA interference; conservation; genotypes; hepatitis B X gene; hepatitis B virus; information content; next-generation sequencing; preS; quasispecies.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Summaries of the proximity function values from the reference sequences to their respective genotypes. Median values correspond to circles (preS) and triangles (5X), while minimal and maximal values are indicated by the lowest and the highest ends of lines crossing circles and triangles, respectively: (a) Base-10 logarithm of minimal, median and maximal lowest proximity functions values (Φk2); (b) Base-2 logarithm of minimal, median and maximal ratios between the two lowest proximity function values (Φl2/Φk2). The safety threshold of 2 for classifying a sequence into a given genotype is denoted by a dotted black line in the graph.
Figure 2
Figure 2
Boxplots representing the distribution of the percentages of the length of preS haplotype sequences affected by gaps, according to their hepatitis B virus genotype. Only the haplotypes from a baseline sample from each patient were included within these boxplots. The statistically significant p-values are obtained after applying a multiple pairwise comparison test (post-hoc Dunn test with Bonferroni correction) and are reported as asterisks: ** p < 0.01 and **** p < 0.0001.
Figure 3
Figure 3
Main HBV genotype in the 125 patients analyzed: (a) Percentage of patients per main genotype; (b) Distribution of ethnicities among patients with the same main genotype. Data on ethnicity was not available for 7 patients (reported in orange).
Figure 4
Figure 4
Analysis of conservation in the 144-nucleotide preS fragment: (a) Localization of the most conserved regions, with the genotype-specific positions of each of them. Regions with overlapping positions across all genotypes (A, C, D, and E) are shown in red, and portions shared between two or more regions in different genotypes are framed in grey; (b) Sequence logos of the conserved regions per genotype. Gt, Genotype.
Figure 5
Figure 5
Analysis of conservation in the sequence of the 5X amplicon: (a) Localization of the most conserved regions, with the positions of each of them. Regions with overlapping positions across all genotypes (A, C, D, and E) are depicted in red, and the portions shared between two or more regions in different genotypes are framed in grey; (b) Sequence logos of the first group of pan-genotypic conserved regions per genotype; (c) Sequence logos of the second group of pan-genotypic conserved regions per genotype. Gt, Genotype.
Figure 6
Figure 6
Workflow for amplification of preS and 5X amplicons. The positions in the hepatitis B virus genome of both amplicons are defined by primers preS_M13_Fw and preS_M13_Rv, and 5X_M13_Fw and 5X_M13_Rv, described in the Table 3. The viral genome scheme indicates the regions of the different viral genes overlapped in the sequence of both amplicons: S, depicted in pink, including preS1 and part of preS2 regions in preS; P, shown in blue, including the end of terminal peptide (TP) and most of the spacer coding regions in preS, and most of the RNAse H region (RH) in 5X, which also includes the 5′ end of HBX (X), depicted in purple. The final PCR products, including M13 forward (black continuous lines) and reverse (black dashed lines) sequences, and MID(1-n) (grey ovals) at their ends, were all processed with next-generation sequencing. Additionally, a group of 66 samples, with sufficient volume available after next-generation sequencing, were selected for additional Sanger sequencing.
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