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IJID 1868 1–10 International Journal of Infectious Diseases xxx (2014) e1–e10
Contents lists available at ScienceDirect
International Journal of Infectious Diseases journal homepage: www.elsevier.com/locate/ijid 1 2 3 4 5 6 7 8
Molecular epidemiology and genetic characterization of hepatitis B virus in the Indian subcontinent§ Mohamed Ismail a, Kalaiselvi Sivalingam Puhazhenthi b, Jayashree Sivakumar a, Chundamanil Eapen Eapen c, Rajesh Kannangai a, Priya Abraham a,*
Q1 Ashrafali
a
Departments of Clinical Virology, Christian Medical College, Vellore 632004, Tamil Nadu, India Freelance Software Developer, Chennai, Tamil Nadu, India c Department of Gastrointestinal Sciences and Hepatology, Christian Medical College, Vellore, Tamil Nadu, India b
A R T I C L E I N F O
S U M M A R Y
Article history: Received 17 July 2013 Received in revised form 29 November 2013 Accepted 6 December 2013
Background: Hepatitis B virus (HBV) is a gradually evolving virus. The aim of this study was to characterize the distribution pattern of HBV genotypes and subgenotypes and HBsAg subtypes in chronic hepatitis B subjects from the Indian subcontinent. We also sought to investigate the genetic diversity of HBV genotypes and its influence on the therapeutic response. Methods: A total of 295 chronic hepatitis B subjects were studied. HBV genotypes and subgenotypes were determined using the generated HBV reverse transcriptase (rt) sequences. HBsAg subtypes were predicted using a newly developed automated program in Microsoft Visual Basic (VB6). Genetic diversity was characterized by calculating the mean genetic distance (d), the number of synonymous substitutions per synonymous site (dS), and the number of non-synonymous substitutions per non-synonymous site (dN). The virological response was measured by HBV DNA levels. Results: In southern India, the predominant HBV subgenotype/subtype was D2/ayw3 (79.1%). In eastern India, C1/adr (28.2%) was found to be the predominant subgenotype/subtype, followed by A1/adw2 (25.4%). In the north-eastern region, C2/adr, D2/ayw3, and D5/ayw3 were predominant and were each identified in 20.8% of subjects. In treatment-naı¨ve subjects, the d, dS, and dN of genotype D sequences were higher compared to genotypes C and A. Additionally, the d, dS, and dN of HBV rt sequence were higher in subjects who subsequently showed a virological response to nucleos(t)ide analogues as compared to non-responders, irrespective of the genotypes tested (p = 0.014 to p < 0.0001). Conclusions: We have described the distribution of HBV genotypes and subgenotypes and HBsAg subtypes in three major regions of the Indian subcontinent. HBV genetic diversity may play a pivotal role in the clinical outcome of chronic hepatitis B. ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. All rights reserved.
Corresponding Editor: Larry Lutwick, Kalamazoo, MI, USA Keywords: Hepatitis B virus Genotype Subgenotype Subtype Genetic diversity HBV evolution India
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1. Introduction
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Hepatitis B virus (HBV) is a circular, partially double-stranded DNA virus of the family Hepadnaviridae. HBV replicates through a reverse-transcribed RNA intermediate, and the low fidelity rates of the reverse transcriptase enzyme lead to highly error-prone nucleotide synthesis.1 Hepadnaviruses are known to have originated more than 19 million years ago, and the evolutionary rate of HBV is estimated to be 7.72 10 4 substitutions/site/year.2,3
§ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. * Corresponding author. Tel.: +91 416 2282772; fax: +91 416 2232035. E-mail address:
[email protected] (P. Abraham).
Together with the higher evolutionary rate, selection pressure imprints a high viral genetic diversity. HBV is classified into genotypes and subgenotypes with an intergenotypic diversity of 8% and intragenotypic diversity of 4%, respectively.4 Accordingly, eight major HBV genotypes (A–H), two novel genotypes (I and J), and several subgenotypes are well described.4–7 HBV strains are also categorized into nine major hepatitis B surface antigen (HBsAg) subtypes based on their antigenic determinants in the major hydrophilic region.8 Further extensive investigations may lead to the recognition of yet unidentified HBV variants. HBV genotypes are known to show a geographical pattern in their distribution and have been used to trace the migration of populations from geographically distant regions.7,9 Moreover, HBV genotypes and HBsAg subtypes have been reported to influence disease progression and treatment response.10–13 Therefore, the molecular characterization of HBV strains is important for disease monitoring and clinical outcomes.
1201-9712/$36.00 – see front matter ß 2014 The Authors. Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. All rights reserved. http://dx.doi.org/10.1016/j.ijid.2013.12.007
Please cite this article in press as: Ismail AM, et al. Molecular epidemiology and genetic characterization of hepatitis B virus in the Indian subcontinent. Int J Infect Dis (2014), http://dx.doi.org/10.1016/j.ijid.2013.12.007
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IJID 1868 1–10 e2
A.M. Ismail et al. / International Journal of Infectious Diseases xxx (2014) e1–e10
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Given the importance of HBV classification, we investigated the distribution pattern of HBV genotypes and subgenotypes and HBsAg subtypes in chronic hepatitis B subjects from the Indian subcontinent. We also characterized the genetic diversity between HBV genotypes and its influence on the subsequent therapeutic response.
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2. Materials and methods
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2.1. Study subjects
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A total of 295 subjects with chronic hepatitis B were enrolled in this study. The study subjects comprised individuals attending the liver clinic of a tertiary care hospital in South India. The subjects were recruited between January 2007 and November 2011 and were part of our HBV antiviral resistance mutations study.14,15
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2.2. HBV polymerase/reverse transcriptase gene amplification and sequencing
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The HBV polymerase gene covering the entire reverse transcriptase (rt) region was amplified using Platinum Taq High Fidelity (Invitrogen, Carlsbad, CA, USA). PCR and the bi-directional sequencing reaction were performed as described previously.14 The nucleotide sequences generated in this study have been deposited in the GenBank database under accession numbers GU798963 to GU799059 and JQ514280 to JQ514554.
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2.3. Determination of HBV genotypes
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HBV genotypes were determined using the HBVseq program in the Stanford database.
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2.4. Determination of HBV subgenotypes
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The study sequences were aligned with published sequences representing all known HBV subgenotypes.4,16–19 Multiple sequence alignment was performed using the built-in ClustalW integrated in MEGA4.20 HBV subgenotypes were determined by phylogenetic analysis in MEGA4 using the neighbour-joining method with a bootstrap test of 1000 replicates and maximum composite likelihood algorithm.
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2.5. Determination of HBsAg subtypes
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An automated computer programme for HBsAg subtype determination was developed in Microsoft Visual Basic (VB6). The overlapping surface gene sequence of HBV rt (155–835 nucleotides) was translated to the corresponding surface gene amino acids using the BioEdit tool. The HBsAg subtypes were then determined by the subtype program that examines every combination of amino acids at positions 122, 160, 127, 159, and 140 (in that order), as deduced by Purdy et al.8
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2.6. HBV genetic diversity
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An analysis of the number of base substitutions per site between sequences (genetic distance (d)) was conducted using the maximum composite likelihood method in MEGA4. The number of synonymous substitutions per synonymous site (dS) and the number of non-synonymous substitutions per non-synonymous site (dN) were calculated using the Nei–Gojobori model with Jukes–Cantor correction in MEGA4. The baseline analysis was performed for a total 197 treatment-naive chronic hepatitis B subjects.
2.7. Statistical analysis
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Categorical variables were compared using the Chi-square test. The d, dS, and dN were compared using the Kruskal–Wallis test. A p-value of
