Hepatitis B virus reverse transcriptase sequence variant database for sequence analysis and mutation discovery

Abstract

Drug resistance resulting from reverse transcriptase (RT) mutations is one of the main obstacles to successful hepatitis B virus (HBV) therapy. Indeed, HBV treatment guidelines recommend HBV genotypic resistance testing for patients receiving nucleos(t)ide RT inhibitors (N(t)RTIs) who develop virological failure. N(t)RTI-resistance mutations at 10 RT positions have been well characterized in phenotypic studies, however, data are lacking on the relative frequency of these mutations in N(t)RTI-treated and untreated individuals. There are also few published data on the extent of amino acid variation at most of the 344 positions of HBV RT and the extent to which this variation is influenced by N(t)RTI treatment. We retrieved 23,871 HBV RT sequences from GenBank and reviewed the published reports of these sequences to ascertain the number of individuals from whom the sequences were obtained, the N(t)RTI treatments of these individuals, and the year and region of virus sampling. We then used these data to populate a relational database we named HBVrtDB. As of July 2010, HBVrtDB contained 6811 sequences from 3869 individuals reported in 281 references. Among these 3869 individuals, 73% were N(t)RTI-naïve and 27% received one or more N(t)RTIs. Among the 10 well-characterized N(t)RTI-resistance mutations, L80I/V, V173L, L180M, A181T, T184S, S202G and M204I/V were significantly associated with treatment with lamivudine, an l-nucleoside analog, and A181S/T/V and N236T were significantly associated with treatment with adefovir, an acyclic nucleoside phosphonate. A similar analysis of ten additional less well-characterized resistance mutations demonstrated a significant association with N(t)RTI treatment for four of the mutations: L82M, S85A, A200V, and Q215S. We also created an interactive program, HBVseq, to enable users to identify mutations in submitted sequences and retrieve the prevalence of these mutations in HBVrtDB according to genotype and N(t)RTI treatment. HBVrtDB and HBVseq are available at http://hivdb.stanford.edu/HBV/releaseNotes/.

Figure 1. HBV BLAST-Hits DB and HBVrtDB Schemas

HBVrtDB was seeded with a blast search of the GenBank viral sequence files using an HBV RT amino acid sequence. Filtered BLAST hits were aggregated by the GenBank reference field and parsed to create a relational database, HBV BLAST-Hits DB (Figure1A). Each set of sequences from a reference was annotated and the resulting annotation was used to populate the tables in HBVrtDB (Figure1B).

Figure 2. Prevalence of Well-Characterized Drug Resistance Mutations in HBVrtDB According To Genotype and Treatment

A schematic representation of the type of data produced by HBVseq or available in the “HBV RT Mutations According to Genotype and Treatment” page. This figure shows ten rows of data corresponding to the ten most widely recognized HBV N(t)RTI-resistance positions (Keeffe et al., 2008b; Lok et al., 2007). The first column indicates the RT position and columns 2 through 10 indicate the percent prevalence of mutation at these 10 positions in N(t)RTI-naïve individuals according to virus genotype. Column 11 contains the pooled (across all genotypes) percent prevalence of mutations in N(t)RTI-naïve individuals. Column 12 contains the percent prevalence of each mutation in individuals who received L-nucleosides and/or ETV but no acyclic nucleoside phosphonates (ANPs). Column 13 contains the percent prevalence of each mutation in individuals who received ANPs but no L-nucleosides or ETV. Column 14 contains the percent prevalence of each mutation in individuals who received L-nucleoside and/or ETV as well as one or more ANPs. In each column, the consensus amino acid is shown at the top of each cell and reported variants along with their percent prevalence (shown as superscripts) are indicated below the consensus. Each mutation represents a hyperlink to the references in which the mutation was reported. Mutations significantly associated with L-nucleosides and/or ETV (column 12) or ANPs (column 13) are shown in bold.

Figure 3. Prevalence of Less Well-Characterized Drug Resistance Mutations in HBVrtDB According To Genotype and Treatment

A schematic representation of the type of data produced by HBVseq or available in the “HBV RT Mutations According to Genotype and Treatment” page. This figure shows ten rows of data corresponding to the ten less well-characterized drug-resistance mutations (see text). The first column indicates the RT position and columns 2 through 10 indicate the percent prevalence of mutation at these 10 positions in N(t)RTI-naïve individuals according to virus genotype. Column 11 contains the pooled (across all genotypes) percent prevalence of mutations in N(t)RTI-naïve individuals. Column 12 contains the percent prevalence of each mutation in individuals who received L-nucleosides and/or ETV but no acyclic nucleoside phosphonates (ANPs). Column 13 contains the percent prevalence of each mutation in individuals who received ANPs but no L-nucleosides or ETV. Column 14 contains the percent prevalence of each mutation in individuals who received L-nucleoside and/or ETV as well as one or more ANPs. In each column, the consensus amino acid is shown at the top of each cell and reported variants along with their percent prevalence (shown as superscripts) are indicated below the consensus. Each mutation represents a hyperlink to the references in which the mutation was reported. Mutations significantly associated with L-nucleosides and/or ETV (column 12) or ANPs (column 13) are shown in bold.

Figure 4. The HBVseq User Interface

The HBVseq user interface including the sequence submission form (Figure 4A); program output showing the genotype of the submitted sequence, the established drug resistance RT mutations identified in the sequence and a table showing the percent prevalence of each identified mutation in HBVrtDB according to genotype and nucleos(t)ide RT inhibitor (N(t)RTI) treatment (Figure 4B); and the detailed sequence information page accessible through each mutation's hyperlink (Figure 4C). The example shown here is a genotype G sequence containing a well-characterized drug resistance mutation V173L. The circled text in (Figure 4B) shows the percent prevalence of V173L in L-nucleoside and/or ETV-treated individuals. By clicking the V173L hyperlink (Figure 4B), the list of published reports of V173L is shown on a new page (Figure 4C).