# HBVdb: a knowledge database for Hepatitis B Virus

Juliette Hayer, Fanny Jadeau, G. Deleage, Alan Kay, Fabien Zoulim, Christophe Combet

► To cite this version:

Juliette Hayer, Fanny Jadeau, G. Deleage, Alan Kay, Fabien Zoulim, et al.. HBVdb: a knowledge database for Hepatitis B Virus. Nucleic Acids Research, 2012, 41 (D1), pp.D566-D570. 10.1093/nar/gks1022. hal-04406497

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# HBVdb: a knowledge database for Hepatitis B Virus

Juliette Hayer¹, Fanny Jadeau¹, Gilbert Deléage¹, Alan Kay², Fabien Zoulim²,³ and Christophe Combet¹,*

¹Unité Bases Moléculaires et Structurales des Systèmes Infectieux; UMR 5086 CNRS - Université Claude Bernard Lyon 1; IBCP FR 3302 - 7, passage du Vercors, 69367 Lyon CEDEX 07, ²INSERM, U1052, Viral Hepatitis Research Laboratory; Université Lyon 1, 151, cours Albert Thomas, 69003 Lyon, and ³Hospices Civils de Lyon, Hepatology Department, 69004 Lyon, France

Received August 14, 2012; Revised September 28, 2012; Accepted October 3, 2012

## ABSTRACT

We have developed a specialized database, HBVdb (http://hbvdb.ibcp.fr), allowing the researchers to investigate the genetic variability of Hepatitis B Virus (HBV) and viral resistance to treatment. HBV is a major health problem worldwide with more than 350 million individuals being chronically infected. HBV is an enveloped DNA virus that replicates by reverse transcription of an RNA intermediate. HBV genome is optimized, being circular and encoding four overlapping reading frames. Indeed, each nucleotide of the genome takes part in the coding of at least one protein. However, HBV shows some genome variability leading to at least eight different genotypes and recombinant forms. The main drugs used to treat infected patients are nucleos(t)ides analogs (reverse transcriptase inhibitors). Unfortunately, HBV mutants resistant to these drugs may be selected and be responsible for treatment failure. HBVdb contains a collection of computer-annotated sequences based on manually annotated reference genomes. The database can be accessed through a web interface that allows static and dynamic queries and offers integrated generic sequence analysis tools and specialized analysis tools (e.g. annotation, genotyping, drug resistance profiling).

## INTRODUCTION

Hepatitis B virus (HBV) is a major health problem worldwide with more than 350 million people being chronic carriers. Chronic HBV infection is associated with a significantly increased risk of developing severe liver diseases, including liver cirrhosis, and hepatocellular carcinoma (HCC), one of the most common forms of human cancer. The estimated risk of HCC in chronic HBV carriers is ~100 times greater than in uninfected individuals (1).

Currently available anti-HBV drugs have limitations. Indeed, interferon alpha administration is associated with adverse reactions, while nucleos(t)ide analogs are virostatic and require long-term administration (2,3).

HBV is an enveloped DNA virus that belongs to the *Hepadnaviridae*, a family of hepatotropic DNA viruses infecting certain mammalian or avian hosts (4). It contains a small (~3.2 kb), partially double-stranded relaxed-circular DNA (rcDNA) genome that replicates by reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). The genome encodes four overlapping open reading frames (ORFs) that are translated to produce the viral core protein (5,6), the surface proteins (5,7), a polymerase/reverse transcriptase (RT) (2,4), and HBx (8,9). The HBV life cycle starts with the binding of the virus to an unknown receptor of the host cell. Then, the viral particle is internalized. The virion rcDNA is delivered to the nucleus, where it is repaired to form a covalently closed-circular cccDNA. The episomal cccDNA serves as the template for the transcription of the pgRNA and the other viral mRNAs by the host RNA polymerase II (10).

The viral genome is variable because of the spontaneous error rate of the viral polymerase and the lack of proof reading activity. There are eight genotypes of HBV designated A-H based on >8% nt variation over the entire genome. The eight HBV genotypes are distributed in distinct geographical localizations (11–13). Recombinant forms involving different genotypes have also been reported (14). However, the extensive overlap between the four encoded ORFs limits the diversity that the virus can tolerate. Indeed, every nucleotide participates in the coding of at least one viral protein attesting of an optimized small genome (15,16). Moreover, the genome variability is also constrained by environmental pressure exerted by the host immune response and the antiviral drugs for treated patients.

To allow researchers to investigate the genetic variability of HBV sequences and viral resistance to treatment, several databases and repositories (17–19) have been published to date. Moreover, tools specific to HBV genotyping (20–22) are available for virologists, as well

<footnote>*To whom correspondence should be addressed. Tel: +33 4 37 65 29 47; Fax: +33 4 72 72 26 04; Email: christophe.combet@ibcp.fr</footnote>
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Annotation and genotyping processes. (A) The annotation procedure starts with the computation of a pairwise alignment between the query sequence (Q) and the most similar duplicated reference genome sequence (R1, R2: replicate 1, 2; nn indicates missing nucleotides between shifted and fixed parts of the query sequence). This alignment is split up into CDS alignments that are optimized before the mapping of features and the transfer of annotations. (B) Genotyping process. These pairwise alignments between query (Q) and reference sequences (e.g. GenoA, genotype A reference genome) are iterated using sliding windows to compute a matrix of mean identities. The matrix is used to produce the array of genotype at each query sequence position. Predicted genotype is deduced from this array.</figcaption></figure>

## DATABASE CONTENT

The text format of an HBVdb entry is an extension of the ENA-Annotation format as used for the euHCVdb (34). Some elements of the ENA-Annotation entry are conserved such as the accession number, the organism name, the creation date, and the references. After the annotation procedure, some elements are corrected and/or completed in the entry, mainly in the features. Indeed, a set of new qualifiers that store specific data is added to some features. The qualifier *PRABI_genotype* is added to the *source* feature to indicate a provisional genotype predicted by the genotyping tool. The qualifier *PRABI_name* is added to each feature to ensure standard names across all the database entries. Concerning the protein annotations, some qualifiers are added to the *mat_peptide* features. These qualifiers, noted *PRABI_prodf*, follow the feature table format of the UniProtKB database (35). The *PRABI_prodf* qualifiers describe the protein chains, the protein domains, some sites like active sites (*act_site*) that designate the catalytic residues of enzymes, and the resistance mutations (*res_mutex*) with the drug and the resistance status.

## WEB INTERFACE

The HBVdb is accessible through a website (Figure 2A) divided into two parts. In the static part, the user can find general information about HBV and the nomenclature used through genome organization, protein descriptions (Figure 2B), the reference genomes and the genotypes. The user can also access pre-computed 'Nucleotide' and 'Protein' datasets sorted according to the genotype (rows) and the protein or CDS names (columns). The datasets provide full-length sequences in Pearson/Fasta format, their multiple sequence alignments computed with Muscle (36) and displayed in Clustal W format (Figure 2C), and the corresponding residue repertoires with Shannon entropies that are useful for analysing conserved/variable alignment positions. The user can download the corresponding files for further analysis. Furthermore, the alignments can be interactively edited with the 'EditAlignment' applet developed by our team. In the dynamic part, users can extract their own dataset by combining multiple criteria (e.g. genotype and sequence length and protein name). The datasets can be exported as Pearson/Fasta sequences, accession number lists and entry flat files for further analysis with the integrated analysis tools.

The available analysis tools are either generic or specialized. The generic analysis tools (e.g. BLAST (37) or Clustal W (38)) are available through the NPS@ server (39), that is an integrated sequence analysis web server. 'Annotate' (Figure 2D), 'Genotype' and 'Resistance' (Figure 2E) specialized tools allow the analysis of one or several HBV sequences uploaded by the user. The annotation of a nucleotide sequence produces a result page listing the CDS found in the query sequence, presenting the predicted genotype, and giving the drug resistance status (with a link to resistance output file) if the nucleotide sequence contains the CDS of the RT domain. The results page also gives access to the global pairwise alignment between the query sequence and the reference genome, as well as the pairwise alignments for each CDS. The user can also access the text entry format of the annotated sequence. The annotation of a protein sequence ends up with a result page indicating the most similar sequence used for its annotation, with links to the entry and the pairwise alignment, and the resistance status if the sequence contains the RT domain. The 'Genotype' tool allows the user to genotype nucleotide sequences. It produces a result page giving the predicted genotype, and the genotype computed at each position of the query sequence. The 'Resistance' tool enables the detection of known drug resistance mutations from nucleotide or protein sequences. The output lists the drug, the resistance status (R, I, S) or 'n.a.' if the query sequence does not contain the HBV RT domain, and the identified mutations.

## STATISTICS

HBVdb is available since June 2012. The release 4 (September 2012) comprises 39289 sequences, including 3606 complete genomes.
<figure><img src="image_3.png" /><figcaption>**Figure 2.** Snapshot of the HBVdb web interface. (A) The HBVdb homepage, with the menu bar and all the menus repeated as buttons for ease-of-use. (B) The HBV core protein information page. (C) The multiple sequence alignment of the genotype H SHBs proteins. (D) The *Annotate* tool detailed result page for an input nucleotide sequence showing the CDS positions with links to their alignment, the genotype and the resistance status. (E) The *Resistance* tool result page showing the resistance status and the resistance profile.</figcaption></figure>

## CONCLUSIONS AND PERSPECTIVES

HBVdb is a collection of human HBV sequences, annotated thanks to a computer-automated system. The automatic annotation process used to generate HBVdb entries guarantees standard annotations and allows efficient keyword searches. The HBVdb website allows researchers to access pre-computed datasets and to extract data thanks to a dynamic query system. The extracted data can be further analysed with a set of bioinformatics tools available in the NPS@ server. HBVdb website also enables users to annotate their own sequences through the HBVdb automatic annotation process. In the future, the annotation will be enriched with new data (e.g. mapping of promoters) and analysis tools (e.g. ADAPVEM detection) will be added. The HBVdb and related programs will be useful to face the analysis of the flood of sequences that are generated thanks to the Next-Generation Sequencing (NGS) technologies.

## SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online: Supplementary Tables 1 and 2.

## ACKNOWLEDGEMENTS

We acknowledge members of the French ANRS hepatitis network for stimulating discussions through the last years in order to make HBVdb a useful tool for the researchers.

## FUNDING

J.H. is recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche. HBVdb is hosted on the Pôle Rhône-Alpes de BioInformatique platform funded by the Groupement d'Intérêt Scientifique Infrastructures en Biologie Sante et Agronomie (GIS IBiSA). Funding for open access
charge: Groupement d'Intérêt Scientifique Infrastructures en Biologie Sante et Agronomie (GIS IBiSA).

*Conflict of interest statement.* None declared.

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