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Research Note

Analysis of the complete genome of hepatitis B virus subgenotype C2 isolate NHB17965 from a patient with uncomplicated chronicity

[version 1; peer review: 1 approved, 1 not approved]
PUBLISHED 09 Jul 2018
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Abstract

The number of chronic cases of hepatitis B virus (HBV) is increasing rapidly in the world. Herein, we report a complete genome of HBV subgenotype C2 (HBV/C2) with current common amino acid substitutions from a patient with chronic HBV without liver complications. Complete genome analysis revealed that the isolated strain was a non-recombinant wild type and had several regular substitutions in the reverse transcriptase domain and small surface proteins of HBV. The isolated complete sequence could be considered as a chronic reference strain of HBV/C2 in Bangladesh. This study may help clinicians and scientists gain in-depth knowledge on common substitutions of HBV/C2 genome and to identify potential therapies against chronic HBV infections.

Keywords

HBV/C2, Chronic, Non-recombinant, Bangladesh

Introduction

The number of cases of chronic liver disease caused by hepatitis B virus (HBV) are increasing markedly1. Globally, more than 2 billion people have been infected by HBV2 and, according to the World Health Organization (WHO) approximately 257 million were living with HBV in 2017. In Bangladesh, the rate of HBV chronicity is 2–6%3, which makes it relatively higher risk than other infectious diseases.

HBV genome comprises a partially double-stranded covalently closed circular DNA that encodes four highly overlapping major open reading frames4. Due to the absence of proof-reading activity, the mutation rate of HBV is high5; hence, recombinant strains are evolving with a common pattern. Most of the HBV cases are chronic, which has a high possibility of causing liver cirrhosis6 and hepatocellular carcinoma7. In Bangladesh, there are no reported reference complete sequence of HBV chronic strain of subgenotype C2. Hence, we isolated the complete genome of a HBV/C2 strain collected from a patient without liver complications, though carrying the virus for a long time.

Methods

Isolation and sequencing

An HBV-positive plasma sample was collected from a 45-year-old male patient in a tertiary hospital in Dhaka, Bangladesh after obtaining the patient’s written informed consent. The infected patient had chronic liver disease, as determined by ultrasonography. The patient was diagnosed with chronic HBV infection recently, with a high viral load in the plasma. However, the patient was not showing signs of jaundice, though was affected by fever, nausea, vomiting and fatigue. The study was approved by the Research Ethics Committee of National Institute of Biotechnology, Bangladesh (NIBREC2015-01). The patient was not taking any antiviral therapy and was diagnosed 1 month prior to obtainment of the plasma sample. HBV DNA was extracted from the sample using the QIAamp MinElute Virus Spin kit (Qiagen, Germany). The complete HBV genome was amplified by six sets of primer pairs used previously in another study8 using a conventional PCR method. The primer sequences and their annealing temperatures were as follows: set 1, forward- AAGCTCTGCTAGATCCCAGAGT, reverse- AGTTGGCGAGAAAGTGAAAGCCTG, 56°C; set 2, forward- CCTATTGATTGGAAAGTATGTCA, reverse- AACAGACCAATTTATGCCTA, 48°C; set 3, forward- GAGACCACCGTGAACGCCCA, reverse- CCTGAGTGCTGTATGGTGAGG, 56°C; set 4, forward- TTCACCTCTGCCTAATCATC, reverse- ATAGGGGCATTTGGTGGTCT, 52°C; set 5, forward- TCAGGCAACTATTGTGGTTTCA, reverse- GGGTTGAAGTCCCAATCTGGATT, 51°C; set 6, forward- GGGTCACCATATTCTTGGGAA, reverse- CGAGTCTAGACTCTGTGGTA, 51°C. For a mixture of 25 µl reaction volume, 12.5 µl of 2X MasterMix (Thermo Fisher Scientific, USA), 1 µl each of forward and reverse primers (IDT, USA), 9.5 µl of nuclease-free water (Thermo Fisher Scientific, USA) and 2 µl of template DNA were used. The condition of the PCR reaction was 1 cycle at 95°C for 10 min, 35 cycles at 95°C for 1 min, with the aforementioned annealing temperatures for 1 min and 72°C for 1 min, and a final cycle for 10 min at 72°C. Sanger sequencing was performed using the BigDye Terminator version 3.1 cycling sequencing kit (Applied Biosystems, USA) by ABI 3130 Genetic Analyser (SeqGen, CA, USA) and by thermal cycler (Sigma-Aldrich, Germany) using the described annealing temperatures as per manufacturer’s instructions after the purification of PCR products using PureLink PCR Purification Kit (Thermo Fisher Scientific, USA), performed in accordance with the manufacturer’s protocol. Next, the sequenced contigs were assembled using the Seqman tool of DNASTAR Lasergene version 7.29.

Analysis

The subgenotyping and mutation analysis of the sequenced genome were performed using the HBV Geno2Pheno tool version 2 using the default parameters, comparing against the HBV genotype D consensus sequence. Recombination analysis of the sequence was performed using the NCBI genotyping tool. The complete genome was deposited in the GenBank under the accession number MH220971.

Results and discussion

Analysis of the complete genome denotes that the isolate studied here, termed NHB17965, comprises HBV genotype C and subgenotype C2 (HBV/C2) with a GC content of 48.77%. Recombination analysis using the NCBI Genotyping tool showed that NHB17965 is a non-recombinant wild-type HBV isolate (Figure 1).

7c495a30-dd52-44a0-b584-f6c15937d4dd_figure1.gif

Figure 1. Recombination analysis of the isolate NHB17965.

The Simplot diagram was generated using the NCBI Genotyping tool.

Isolate NHB17965 was observed to have amino acid substitutions H9Y, N13H, I91L, P109S, T128N, I269L and V278I in the polymerase domain and S53L, P120T, I126T and S210N in the small hepatitis B surface protein as analysed by HBV Geno2Pheno tool, compared against the HBV genotype D consensus sequence. These substitutions may be the results of regular genomic changes to HBV because of a lack of proof-reading activity of the viral reverse transcriptase, and may not signify any danger. Hence, the isolate NHB17965 could be considered as a reference strain of chronic HBV/C2 infection in Bangladesh.

The findings of this study will help clinicians and scientists to gain substantial knowledge about the current common genomic substitutions of HBV/C2 and to develop treatments against chronic HBV infections.

Data availability

Genome of the HBV strain isolated in this study, MH220971.

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Shaha M, Sarker PK, Hossain MS et al. Analysis of the complete genome of hepatitis B virus subgenotype C2 isolate NHB17965 from a patient with uncomplicated chronicity [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2018, 7:1023 (https://doi.org/10.12688/f1000research.15090.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Open Peer Review

Current Reviewer Status: ?
Key to Reviewer Statuses VIEW
ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
Version 1
VERSION 1
PUBLISHED 09 Jul 2018
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29
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Reviewer Report 07 Aug 2018
Paul Klapper, University of Manchester, Manchester, UK 
Not Approved
VIEWS 29
This paper reports a sequence analysis of a strain of hepatitis B virus. However, there are some aspects of the paper that merit attention. In the Abstract and again in the Introduction the authors state: The number of chronic cases of hepatitis ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Klapper P. Reviewer Report For: Analysis of the complete genome of hepatitis B virus subgenotype C2 isolate NHB17965 from a patient with uncomplicated chronicity [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2018, 7:1023 (https://doi.org/10.5256/f1000research.16434.r36692)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 13 Aug 2018
    Modhusudon Shaha, Microbial Biotechnology Division, National Institute of Biotechnology, Dhaka, 1349, Bangladesh
    13 Aug 2018
    Author Response
    We would like to thank the reviewer for his constructive comments on the manuscript. Herein, the responses to the comments are given.
     
    ‘The number of chronic cases of hepatitis ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 13 Aug 2018
    Modhusudon Shaha, Microbial Biotechnology Division, National Institute of Biotechnology, Dhaka, 1349, Bangladesh
    13 Aug 2018
    Author Response
    We would like to thank the reviewer for his constructive comments on the manuscript. Herein, the responses to the comments are given.
     
    ‘The number of chronic cases of hepatitis ... Continue reading
Views
22
Cite
Reviewer Report 23 Jul 2018
Mohammad Ariful Islam, Jagannath University, Dhaka, Bangladesh 
Approved
VIEWS 22
The overall level of the paper is good: even if it is quite simple, it is well written and some important considerations are highlighted.

The manuscript talks about the complete genome analysis of hepatitis B virus and ... Continue reading
CITE
CITE
HOW TO CITE THIS REPORT
Islam MA. Reviewer Report For: Analysis of the complete genome of hepatitis B virus subgenotype C2 isolate NHB17965 from a patient with uncomplicated chronicity [version 1; peer review: 1 approved, 1 not approved]. F1000Research 2018, 7:1023 (https://doi.org/10.5256/f1000research.16434.r35849)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
  • Author Response 01 Aug 2018
    Modhusudon Shaha, Microbial Biotechnology Division, National Institute of Biotechnology, Dhaka, 1349, Bangladesh
    01 Aug 2018
    Author Response
    We would like to thank the reviewer for his constructive comments on the manuscript.

    NHB17965 indicates the sample identification number given by the laboratory. 

    There are no reference sequences used in this ... Continue reading
COMMENTS ON THIS REPORT
  • Author Response 01 Aug 2018
    Modhusudon Shaha, Microbial Biotechnology Division, National Institute of Biotechnology, Dhaka, 1349, Bangladesh
    01 Aug 2018
    Author Response
    We would like to thank the reviewer for his constructive comments on the manuscript.

    NHB17965 indicates the sample identification number given by the laboratory. 

    There are no reference sequences used in this ... Continue reading

Comments on this article Comments (0)

Version 3
VERSION 3 PUBLISHED 09 Jul 2018
Comment
Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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