Volume 6 Supplement 1

Proceedings of the 2011 International Conference on Bioinformatics and Computational Biology (BIOCOMP'11)

Open Access

Development of a novel DNA sequencing method not only for hepatitis B virus genotyping but also for drug resistant mutation detection

Contributed equally
BMC Medical Genomics20136(Suppl 1):S15

DOI: 10.1186/1755-8794-6-S1-S15

Published: 23 January 2013

Abstract

Background

In HBV-infected patients, different genotypes of the hepatitis B virus influence liver disease progression and response to antiviral therapy. Moreover, long-term antiviral therapy will eventually select for drug-resistant mutants. Detection of mutations associated to antiviral therapy and HBV genotyping are essential for monitoring treatment of chronic hepatitis B patients.

Results

In this study, a simple method of partial-S gene sequencing using a common PCR amplification was established for genotyping clinical HBV isolates sensitively, which could detect the drug-resistant mutations successfully at the same time.

Conclusions

The partial S gene sequencing assay developed in this study has potential for application in HBV genotyping and drug resistant mutation detection. It is simpler and more convenient than traditional S gene sequencing, but has nearly the same sensitivity and specificity when compared to S gene sequencing.

Background

Eight distinct genotypes (A to H) of hepatitis B virus (HBV) have been identified, and this classification is based on the distance of the nucleotide sequence from the viral genome of 8% or greater [1, 2]. These genotypes also have a distinct geographical distribution, while genotypes B and C are more common in China. Since genotypes of HBV influence liver disease progression and response to antiviral therapy in HBV-infected patients, several methods have been developed for genotyping of HBV strains [3], these include sequence analysis; microarray (DNA-Chip) [4, 5]; reverse hybridization [6]; restriction fragment length polymorphism (RFLP) [7]; serological assays and genotype-specific PCR assays [8, 9]. These techniques have the disadvantage that they are based on specific hybridization of HBV DNA, and nucleotide changes can interfere with this process and subsequent sequence analysis. For example, RFLP and multiple PCR methods might give wrong results even for a single base mutation. Serological assay has a low cost and does not rely on PCR amplification, but it is still subjected to the effects of specific base mutation. INNO-LiPA HBV genotyping assay has the limitation of high cost, and also its likelihood to be affected by the specific binding site mutant gene [10, 14]. Sequence analysis is definitely the most accurate method and not subject to these constraints, but it is also the most labor intensive technique and needs nested PCR to increase the sensitivity.

As we all know, there are several antiviral therapies--such as interferon; pegylated interferon or nucleotide/nucleoside analogs--widely used to treat HBV infection. None of these therapies can eradicate HBV infection and all often induce drug-resistant mutants. As a result, HBV genotyping and the detection of mutations that confer drug resistance help select an appropriate treatment strategy and monitor the treatment. However, there are a limited number of methods that enable simultaneous genotyping and mutation detection. In this study, we used the partial S-gene sequencing using common PCR to genotype HBV, which is simpler and more sensitive compared with the S-gene sequencing.

Partial S-gene sequencing means sequencing part of the S gene from 370 nt to 861 nt. This part of the S gene we chose is relatively conserved and has many drug-resistant mutant sites, so it could be used in both HBV genotyping and in analysis of HBV drug resistant mutation.

Results and discussion

Phylogenetic tree analysis

First, phylogenetic tree analysis was used for testing the possibility of HBV genotyping using partial S gene sequencing. Reference sequences from 32 HBV genomes of eight different genotypes were used (shown as Table 1). Software MEGA4 was used to analyze these genomes to get the phylogenetic trees of genome sequencing; S gene sequencing and partial S gene sequencing respectively. All these sequences could be genotyped successfully by partial S gene sequencing. The results of phylogenetic tree analysis indicated that the partial S gene sequencing had nearly the same phylogenetic tree as that of the S gene sequencing (shown as Figure 1, 2, 3).
Table 1

Reference sequences for genotyping

Genotype A

AM282986

gi_59418

gi_1155012

gi_15419837

gi_5114084

Genotype B

gi_21280301

gi_221497

gi_221498

gi_4323201

gi_6063442

Genotype C

gi_13365548

gi_22415734

gi_6063452

NC_003977

gi_3582357

Genotype D

gi_329640

gi_736003

gi_329667

gi_62280

gi_59439

Genotype E

gi_452617

gi_6691492

   

Genotype F

gi_11191875

gi_59422

gi_12247041

gi_452637

 

Genotype G

gi_18146661

gi_6983934

gi_19849032

  

Genotype H

gi_22135696

gi_22135711

gi_22135726

  
https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig1_HTML.jpg
Figure 1

Phylogenetic tree map of HBV genome sequencing. 32 HBV genomes of eight different genotypes were sequenced by HBV genome sequencing, and software MEGA4 was used to analyze these genomes to get the phylogenetic trees of genome sequencing. Tree Inference: [Method: Neighbor-Joining; Phylogeny Test and options: Bootstrap (1000 replicates; seed = 100000)]; Include Sites: [Gaps/Missing Data: Complete Deletion]; Substitution Model: [Model: Nucleotide: Maximum Composite Likelihood; Substitutions to Include: d: Transitions + Transversions; Pattern among Lineages: Same (Homogeneous); Rates among sites: Uniform rates]

https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig2_HTML.jpg
Figure 2

Phylogenetic tree map of HBV S gene sequencing. 32 HBV genomes of eight different genotypes were sequenced by HBV S gene sequencing, and software MEGA4 was used to analyze these genomes to get the phylogenetic trees of HBV S gene sequencing. Tree Inference: [Method: Neighbor-Joining; Phylogeny Test and options: Bootstrap (1000 replicates; seed = 100000)]; Include Sites: [Gaps/Missing Data: Complete Deletion; Codon Positions: 1st+2nd+3rd]; Substitution Model: [Model: Nucleotide: Maximum Composite Likelihood; Substitutions to Include: d: Transitions + Transversions; Pattern among Lineages: Same (Homogeneous); Rates among sites: Uniform rates]

https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig3_HTML.jpg
Figure 3

Phylogenetic tree map of HBV partial S gene sequencing. 32 HBV genomes of eight different genotypes were sequenced by HBV partial S gene sequencing, and software MEGA4 was used to analyze these genomes to get the phylogenetic trees of HBV partial S gene sequencing. Tree Inference: [Method: Neighbor-Joining; Phylogeny Test and options: Bootstrap (1000 replicates; seed = 100000)]; Include Sites: [Gaps/Missing Data: Complete Deletion; Codon Positions: 1st+2nd+3rd]; Substitution Model: [Model: Nucleotide: Maximum Composite Likelihood; Substitutions to Include: d: Transitions + Transversions; Pattern among Lineages: Same (Homogeneous); Rates among sites: Uniform rates]

To further evaluate the effect of partial S gene sequencing, 53 HBV genotype A samples; 43 HBV genotype B samples; 50 HBV genotype C samples and 51 HBV genotype D samples were genotyped using partial S gene sequencing and S gene sequencing respectively (shown as Table 2). Compared the results of these two methods, we found that the results of partial S gene sequencing are consistent with S gene sequencing, except one sample, EU939630. From further recombinant analysis, we found that EU939630 was a C/B recombinant strain (shown as Figure 4).
Table 2

Sequences for genotyping verification

Genotype A

AB014370

AB330372

AB453986

AJ627227

AM295800

EU594388

 

AB126580

AB330373

AB453987

AJ627228

AM410963

EU594389

 

AB194950

AB453979

AB453988

AM184125

AM494718

EU594390

 

AB194951

AB453980

AB453989

AM184126

AP007263

EU594391

 

AB194952

AB453981

AJ309369

AM282986

EU594383

EU594392

 

AB205118

AB453982

AJ309370

AM295795

EU594384

EU594393

 

AB241114

AB453983

AJ309371

AM295797

EU594385

EU594394

 

AB241115

AB453984

AJ344115

AM295798

EU594386

EU594395

 

AB330371

AB453985

AJ627226

AM295799

EU594387

 

Genotype B

AB014366

AB205119

AB287317

AB287326

EF473977

EU939628

 

AB033554

AB205120

AB287318

AB287327

EU595030

EU939629

 

AB033555

AB205121

AB287319

AB287328

EU595031

EU939630

 

AB115551

AB205122

AB287320

AB287329

EU796066

 
 

AB117759

AB241117

AB287321

AB365445

EU796067

 
 

AB195933

AB287314

AB287322

AB368295

EU796068

 
 

AB195934

AB287315

AB287323

AJ627225

EU796071

 
 

AB195935

AB287316

AB287325

EF473976

EU939627

 

Genotype C

AB014360

AB014374

AB014384

AB026811

AB033557

AB112065

 

AB014362

AB014376

AB014385

AB026812

AB042282

AB112066

 

AB014363

AB014377

AB014389

AB026813

AB042283

AB112348

 

AB014364

AB014378

AB014391

AB026814

AB042284

AB112471

 

AB014365

AB014379

AB014392

AB033550

AB042285

AB112472

 

AB014367

AB014380

AB014393

AB033551

AB105172

 
 

AB014369

AB014381

AB014394

AB033552

AB105173

 
 

AB014371

AB014382

AB014396

AB033553

AB105174

 
 

AB014372

AB014383

AB014399

AB033556

AB112063

 

Genotype D

AB033558

AB109475

AB119253

AB188244

AB210822

EU594431

 

AB033559

AB109476

AB119254

AB188245

EU594422

EU594432

 

AB090268

AB109477

AB119255

AB205126

EU594423

EU594433

 

AB090269

AB109478

AB119256

AB205127

EU594424

EU594434

 

AB090270

AB109479

AB120308

AB205128

EU594425

EU594435

 

AB104709

AB110075

AB126581

AB210818

EU594426

EU594436

 

AB104710

AB116266

AB188241

AB210819

EU594427

 
 

AB104711

AB119251

AB188242

AB210820

EU594428

 
 

AB104712

AB119252

AB188243

AB210821

EU594430

 
https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig4_HTML.jpg
Figure 4

Genotype and recombinant analysis results of EU939630. A: Genotype results of 43 HBV genotype B samples using partial S gene sequencing, 42 samples of which had been genotyped correctly except one, EU939630. The arrow indicates EU939630 was genotyped as HBV genotype C by partial S gene sequencing. B: Recombinant analysis result of EU939630. From the recombinant analysis, we found that EU939630 was a C/B recombinant strain.

Sensitivity of partial S gene sequencing

147 HBV-positive (HBV copies were more than 500 copies/ml) serum samples were sequenced by partial S gene sequencing, 2 of which could not to be sequenced, so the sensitivity of our partial S gene sequencing was 98.64%.

Application of partial S gene sequencing in recombinant HBV isolates

Next, we evaluated the application of partial S gene sequencing in HBV recombinant isolates (shown as Figure 5 and 6). 44 recombinant HBV isolates were collected as shown in Table 3. The genotyping results indicated that there were 38 samples with the same results using partial S gene sequencing and S gene sequencing. Other 6 samples had different genotyping results by these two methods, which were isolated from South Africa (2/6); Thailand (1/6) and Vietnam (3/6) respectively. One sample from South Africa (AF297620) failed to genotype by partial S gene sequencing, as there was a recombinant site in its S region (shown as Figure 7) which was analyzed by Simplot software and NCBI Viral genotyping tool.
https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig5_HTML.jpg
Figure 5

Phylogenetic tree map of HBV recombinant strains using S gene sequencing. 44 recombinant HBV isolates were collected to draw the phylogenetic tree map of HBV S gene sequencing by the method described above. Tree Inference: [Method: Neighbor-Joining; Phylogeny Test and options: Bootstrap (1000 replicates; seed = 100000)]; Include Sites: [Gaps/Missing Data: Complete Deletion]; Substitution Model: [Model: Nucleotide: Maximum Composite Likelihood; Substitutions to Include: d: Transitions + Transversions; Pattern among Lineages: Same (Homogeneous); Rates among sites: Uniform rates]

https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig6_HTML.jpg
Figure 6

Phylogenetic tree map of HBV recombinant strains using partial S gene sequencing. 44 recombinant HBV isolates were collected to draw the phylogenetic tree map of HBV partial S gene sequencing by the method described above. Tree Inference: [Method: Neighbor-Joining; Phylogeny Test and options: Bootstrap (1000 replicates; seed = 100000)]; Include Sites: [Gaps/Missing Data: Complete Deletion]; Substitution Model: [Model: Nucleotide: Maximum Composite Likelihood; Substitutions to Include: d: Transitions + Transversions; Pattern among Lineages: Same (Homogeneous); Rates among sites: Uniform rates]

Table 3

Genotype results of 44 HBV recombinant strains

 

Genotyping method

Source

 

S

partial S

genome

 

EU185780

A

A

D3/A2

Argentina

AB194949

A

A

A (A3/Acmr)/E

Cameroon

AY817509

D

D

C/D

China

AY817510

D

D

C/D

China

AY817511

D

D

C/D

China

AY817512

D

D

C/D

China

AY817513

D

D

C/D

China

AY817514

D

D

C/D

China

AY817515

D

D

C/D

China

AY862860

D

D

C/D

China

AY862861

D

D

C/D

China

AY862862

D

D

C/D

China

AY862863

D

D

C/D

China

AY862864

D

D

C/D

China

DQ478881

D

D

C/D

China

DQ478882

D

D

C/D

China

DQ478883

D

D

C/D

China

DQ478884

D

D

C/D

China

DQ478886

D

D

C/D

China

DQ478887

D

D

C/D

China

DQ478888

D

D

C/D

China

DQ478889

D

D

C/D

China

DQ478890

D

D

C/D

China

DQ478891

D

D

C/D

China

DQ478892

D

D

C/D

China

DQ478893

D

D

C/D

China

DQ478894

D

D

C/D

China

DQ478895

D

D

C/D

China

DQ478896

D

D

C/D

China

DQ478897

D

D

C/D

China

DQ478898

D

D

C/D

China

AY057947

C

C

A/C

China

AY057948

D

D

C/D

China

EF103282

A

A

A/D

India

EF103283

A

A

A/D

India

EF103284

A

A

A/D

India

AB270534

D

D

C/D

Mongolia:Ulaanbaatar

AB270535

D

D

C/D

Mongolia:Ulaanbaatar

AF297619

D

A

A/D

South Africa

AF297620

D

-

A/D

South Africa

DQ078791

C

G

G/C

Thailand

AF241407

D

G

C/A/G/B

Vietnam

AF241408

D

G

C/A/G/B

Vietnam

AF241409

D

G

C/A/G/B

Vietnam

https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig7_HTML.jpg
Figure 7

Recombinant analysis results of AF297620. AF297620 was failed to be genotyped by partial S gene sequencing because it had recombinant sites in partial S gene region.

Drug-resistant mutations analysis

The part of the S gene we chose for sequencing has many drug-resistant mutant sites, which means it had potential for use in analysis of HBV drug resistant mutation. To evaluate this, we analyzed all possible sites of resistance mutations (V521L; A529V; A529T; T532A; S550I; rtL180M; rtM204V/I; N584T and K589E) of 147 HBV-positive serum samples [16]. Through partial S gene sequencing we detected rtM204V mutation in one sample successfully (shown in Figure 8), meaning the partial S gene sequencing could be used in both HBV genotyping and in analysis of HBV drug resistant mutation.
https://static-content.springer.com/image/art%3A10.1186%2F1755-8794-6-S1-S15/MediaObjects/12920_2013_Article_360_Fig8_HTML.jpg
Figure 8

Drug resistant mutation analysis by partial S gene sequencing. Through drug resistant mutation analysis of 147 HBV positive serum samples using partial S gene sequencing, one sample was found to be rtM204V mutation. The arrow indicates the A is replaced by G.

Conclusions

HBV genotyping and the detection of drug resistance mutations are important for monitoring the treatment of chronic hepatitis B, but there are a limited number of methods for the simultaneous detection of HBV genotypes and drug resistance mutations [11, 13]. We have established a partial S gene sequencing method to genotype HBV isolates as well as detect drug resistance mutations at the same time. To testify the sensitivity of our partial S gene sequencing, 147 clinical serum samples were used, and 145 samples could be sequenced successfully by this assay, with a sensitivity of 98.64%.

The part of S gene we chose for sequencing has many drug-resistant mutant sites, and we detected all possible mutant sites in this region of 145 samples and found the rtM204V mutation in one sample. That means the partial S gene sequencing could be used in analysis of HBV drug resistant mutation.

To evaluate the potential of partial S gene sequencing for use in HBV genotyping, the phylogenetic tree analysis was used. From the phylogenetic tree mapping, we found that the partial S gene sequencing had nearly the same phylogenetic tree map to that of the S gene sequencing (shown as Figure 2 and 3). This means partial S gene sequencing has the possibility to be used as a promising method in HBV genotyping. To further demonstrate this, 197 HBV positive serum samples of four different genotypes (A, B, C, and D) were genotyped using partial S gene sequencing and S gene sequencing respectively (Table 2). Although at least eight HBV genotypes have been reported, the major HBV genotypes in China are B and C [12, 15]. Genotypes A and D are found in a very small proportion of Chinese patients, and genotypes E, F, G, and H have not been reported in China. As a result, we only evaluated the performance of partial S gene sequencing assay for genotypes A, B, C, and D in our study. We found the results of partial S gene sequencing were consistent with S gene sequencing, except one sample, EU939630, which was proved to be a C/B recombinant strain. From this, we have proved that this assay could specifically detect mutant and wild-type HBV in clinical serum samples.

Considering that the recombinant strain might disturb genotyping effect, we evaluated the application of partial S gene sequencing in 44 HBV recombinant isolates (Table 3). 43 samples could be genotyped correctly by partial S gene sequencing method, meanwhile, S gene sequencing genotyped 41 samples successfully. That means partial S gene sequencing had the potential to take the place of S gene sequencing in the field of HBV genotyping.

Based on these findings, we could draw the conclusion that the partial S gene sequencing assay developed in this study could be applied in HBV genotyping and drug resistant mutation detection. It might be an ideal choice for HBV genotyping for it is simpler and more convenient than traditional S gene sequencing while it has nearly the same sensitivity and specificity as S gene sequencing.

Methods

Serum samples

Serum samples are collected from four hospitals: the Second People Hospital of Guangdong Province, the First Affiliated Hospital of Guangzhou Medical College, Guangzhou Overseas Chinese Hospital, and Guangzhou Huadu Ren-Ai Hospital. All these serum samples were collected in compliance with the Helsinki Declaration, and all the patients who provided serum samples were voluntary. This study was approved by the Institutional Review Board of Wuhan University. All specimens were sampled from sterile blood vessels (excluding anticoagulant) and stored at -20°C.

Sequencing

HBV DNA was isolated from serum (QIAamp DNA Blood Mini Kit, QIAGEN, Hilden, Germany) according to the kit instructions. A product of 491 base pairs of partial S gene was amplified with the primers F (sense, 5'- TCGCTGGATGTGTCTGCGGCGTTTTAT-3') and R (antisense, 5'- ACCCCATCTTTTTGTTTTGTTAGG-3') using a PCR protocol as follows: 12 min at 95°C, 35 cycles of 1 min at 94°C, 1 min at 52°C, 1 min at 72°C, and a final elongation step of 7 min at 72°C, using the AmpliTaq Gold amplification system.

Phylogenetic tree analysis

To testify the feasibility of the partial S gene sequencing used for HBV genotyping, 32 different genotypes of the HBV (http://lancelot.otago.ac.nz) genome sequence were chosen as the reference sequences (shown in Table 1). Three different DNA sequencing methods were used to genotype serum samples for phylogenetic tree mapping: whole genome sequencing, S gene sequencing and partial S gene sequencing.

Notes

Declarations

Acknowledgements

This article has been published as part of BMC Medical Genomics Volume 6 Supplement 1, 2013: Proceedings of the 2011 International Conference on Bioinformatics and Computational Biology (BIOCOMP'11). The full contents of the supplement are available online at http://www.biomedcentral.com/bmcmedgenomics/supplements/6/S1. Publication of this supplement has been supported by the International Society of Intelligent Biological Medicine.

Authors’ Affiliations

(1)
State Key Laboratory of Virology and College of Life Sciences, Wuhan University
(2)
Guangzhou Kingmed Center for Clinical Laboratory
(3)
Wuhan University of Science and Technology

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Copyright

© Wang et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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