Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G Jack Bee Chook,a Woon Li Teo,b Yun Fong Ngeow,b Kok Keng Tee,c Kee Peng Ng,b Rosmawati Mohameda Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysiaa; Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysiab; Centre of Excellence for Research in AIDS (CERiA), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysiac

H

epatitis B virus (HBV) has a partially double-strandedDNA, 3.2-kb-long genome. The virus replicates through the error-prone reverse transcription of an RNA intermediate (1), leading to a high degree of viral genetic variation. HBV, like RNA viruses, has a high evolutionary rate, 10⫺4 to 10⫺5 substitution per genome per year, due to its error-prone reverse transcription replication (2). At least 9 genotypes (A to I) and one provisional genotype (J) for HBV have been defined on the basis of an intergroup diversity of ⬎8% (3). Genotypes B and C are predominant in the East and Southeast Asia regions. The genetic diversity of HBV genomes has made it a useful tool for investigating outbreaks (4), drug resistance mutations (5, 6), and severe liver disease-related mutations (7) and for evolutionary studies (8). In addition to the high mutation rates, intergenotype recombination is common, accounting for about 30% of HBV genomes in the GenBank database (9). A universal method for obtaining HBV genome sequences is therefore desirable but challenging. The design of highly conserved primer sets across all genotypes is necessary in order to achieve a high success rate in the amplification and sequencing of the HBV genome. In this study, we attempted to establish universally conserved primer sets for HBV genotypes A to G. The primer sets were tested in HBV genotypes A to G and local isolates in Malaysia for amplification and sequencing. They are sensitive and robust and obviate any prior knowledge of HBV genotypes.

High Pure Viral Nucleic Acid kit (Roche, Germany), with slight modification in that 55 ␮l of viral DNA was used for elution instead of 50 ␮l. Primer design and nested PCR. Universal primer sets covering the entire HBV genome were designed for nested PCR amplifications based on 5,154 HBV complete genome sequences obtained from the National Centre for Biotechnology Information (NCBI) Nucleotide Database on December 2012. Accession numbers and alignment of the HBV genomes are provided at the following link: https://www.dropbox.com/s/7rq3xb7 pzomtl97/5154%20HBV%20genomes%20aligned%20using%20mafft .zip?dl⫽0. Sequence alignment was performed using MAFFT 6.849 (10). Conserved regions were identified from the consensus sequence created by BioEdit (11). Primers were designed using Primer Express v3.0.1 (Applied Biosystems, NY). Four outer primer sets were designed: they were 251f-1797r (set 1), 2300f-654r (set 2), 1859f-2835r (set 3), and 1584f2396r (set 4). In the nested PCR assay, six inner primer sets were used to generate six overlapping fragments across the HBV genome from PCR products amplified earlier with the four outer primer sets. These six inner primer sets included 251f-1190r and 595f-1797r from outer primer set 1, 2300f-215r and 2819f-617r from outer primer set 2, 1877f-2835r from outer primer set 3, and 1584f-2331r from outer primer set 4 (Fig. 1). The picture of the HBV genome was created with reference to GenBank accession number NC_003977 by using SnapGene (IL). These six primer sets yielded amplicon sizes ranging 748 to 1,203 bp (Table 1). The PCRs were performed using MyFi 2⫻ premix (Bioline, United Kingdom). Each

Received 8 December 2014 Returned for modification 12 January 2015 Accepted 12 March 2015 Accepted manuscript posted online 18 March 2015

MATERIALS AND METHODS Plasma samples and viral DNA extraction. A total of 126 plasma samples were retrieved from the follow-up patients with chronic hepatitis B in University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. Approval for the study was obtained from the UMMC Medical Ethics Committee, acting by the ethical standards of the Declaration of Helsinki. Informed consent was obtained for all subjects. Viral DNA was extracted from 200 ␮l of plasma according to the manufacturer’s protocol with a

June 2015 Volume 53 Number 6

Citation Chook JB, Teo WL, Ngeow YF, Tee KK, Ng KP, Mohamed R. 2015. Universal primers for detection and sequencing of hepatitis B virus genomes across genotypes A to G. J Clin Microbiol 53:1831–1835. doi:10.1128/JCM.03449-14. Editor: A. M. Caliendo Address correspondence to Rosmawati Mohamed, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.03449-14

Journal of Clinical Microbiology

jcm.asm.org

1831

Downloaded from http://jcm.asm.org/ on May 15, 2015 by Yale University

Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.

Chook et al.

50-␮l reaction mixture contained 25 ␮l of 2⫻ premix with 3 mM MgCl2, 2 ␮l of each 10 ␮M primer, and 5 ␮l (first PCR) or 3 ␮l (second PCR) of DNA template. The PCR thermal profile used was 95°C for 3 min, 97°C for 30 s, annealing temperature (Ta) for 1 min, 72°C for 1 min, 35 cycles in the first round and 31 cycles in the second round, and final extension at 72°C for 10 min. Amplified products were stained with RedSafe (Chembio, United Kingdom) and verified by agarose gel electrophoresis under UV transillumination. Coverage of primers. The percentage of coverage for each primer was calculated based on the retrieved HBV genomes described earlier. The percentage of coverage indicates the number of HBV genomes that match a primer, either fully matched or with one allowed mismatch (not including the first 3 nucleotides [nt] near the 3= end) (Table 2). Assay performance. HBV panels purchased from AcroMetrix (Applied Biosystems, NY) were used to determine the limit of detection of each primer set. Plasma concentrations of 50, 100, and 500 IU/ml were employed during testing, whereby 1 IU/ml is equivalent to Roche 5.82 viral copies/ml or 5.3 viral copies/reaction (if 100% extraction efficiency is assumed). The primer sets were applied to 126 plasma samples of known copy numbers, ranging from 20 to ⬎79,780,000 IU/ml. The viral loads of the samples were determined by Roche Cobas TaqMan assay (Germany). DNA sequencing was performed with an ABI Prism 3730XL DNA analyzer using BigDye terminators (Applied Biosystems, CA). Based on 126 plasma samples, the rate of success of PCR amplification was calculated, whereas the rate of success of sequencing for each sequencing primer set was calculated based on the number of successful sequencing over the total number of successful PCR amplifications. All sequenced viral DNA fragments were assembled using the BioEdit built-in CAP contig assembly program (11). In this study, a chromatogram was considered of good quality if (i) at

1832

jcm.asm.org

least 80% of total amplicon length was sequenced and (ii) the noiseto-signal ratio was estimated to be ⬍5%. Application of overlapping primer sets across an HBV reference panel of genotypes A to G. The universal primer sets were evaluated on the First WHO Reference Panel for Hepatitis B Virus Genotypes, consisting of 15 strains of HBV genotypes A to G (PEI code 5086/08), provided by Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institut (PEI). There were three strains each of genotypes A to D and one each of genotypes E to G. The concentrations of these strains ranged from 3.8 to 6.1 log10 IU/ml. The rates of success of PCR amplification and sequencing were calculated.

RESULTS

Coverage. A total of 5,154 HBV genomes were retrieved, encompassing all currently available genotypes A to J. Four overlapping outer primers for heminested PCR were designed. The working Ta of outer primers 251f-1797r (for sets 1a and 1b), 2300f-654r (for sets 2a and 2b), 1859f2-2835r (for set 3), and 1584f-2396r (for set 4) were 54°C, 52°C, 54°C, and 57°C, respectively. Each primer set had coverage ranging from 95 to 99% for 100% identity and coverage of ⬎98% when allowing one nucleotide mismatch (Table 2). The Ta for each inner primer set was also described. Performance. All six primer sets had a confident limit of detection of at least 100 IU/ml, and 50 IU/ml was even achieved for some primer sets (Table 3). When these primer sets were applied to the 126 plasma samples, at least 96% samples turned out to be positive by PCR amplification. All nonamplifiable products were

Journal of Clinical Microbiology

June 2015 Volume 53 Number 6

Downloaded from http://jcm.asm.org/ on May 15, 2015 by Yale University

FIG 1 Organization of an HBV genome (GenBank accession number NC_003977). Nucleotide positions of overlapping PCR and sequencing primer sets were drawn outside the genome, whereas the flanking region of each primer set was drawn with unfilled arrows inside. Primers 251f-1190r (set 1a) and 595f-1797r (set 1b), 2300f-215r (set 2a) and 2819f-617r (set 2b), 1584f-2331r (set 3), and 1877f-2835r (set 4) are shown.

Universal Primers for Sequencing of HBV Genomes

TABLE 1 Primers used for the amplification of HBV complete genomes Sequencing primer

251–269 1190–1174

940

Yes Yes

CAC HTG TAT TCC CAT CCC A CCA ATT TMT GCY TAC AGC CTC AYG CAA CCC CCA CTG G

595–613 1797–1777 1190–1205

1,203

Yes No Yes

Set 2a (Ta ⫽ 50°C) 2300f 215r

CCA CMW AAT GCC CCT ATC AGR AAM ACM CCG CCT GT

2300–2317 215–200

1,131

Yes Yes

Set 2b (Ta ⫽ 50°C) 2819f 617rc 654r

ACC WTA TWC YTG GGA ACA A GAY GAY GGG ATG GGA ATA CA GSC CCA MBC CCA TAG G

2819–2837 617–598 652–637

1,032

Yes Yes No

Set 3 (Ta ⫽ 52°C) 1859f 1877f 2835r

ACT NTT CAA GCC TCC RAG CTG CTG TGC CTT GGR TGG CTT GTT CCC AVG WAT AWG GTG AYC C

1859–1879 1894–1877 2835–2814

959

No Yes Yes

Set 4 (Ta ⫽ 57°C) 1584f 2331r 2396r

ACT TCG MBT CAC CTC TGC ACG T GGA AGY GTK GAY ARG ATA GGG GCA TT GTC KGC GAG GYG AGG GAG TT

1583–1604 2331–2306 2396–2377

748

No Yes No

Sequence (5=–3=)

Position (nt)b

Set 1a (Ta ⫽ 54°C) 251f 1190r

GAC TYG TGG TGG ACT TCT C TCA GCA AAY ACT YGG CA

Set 1b (Ta ⫽ 54°C) 595f 1797r 1190f

a

Suffix “f” indicates forward primer, whereas suffix “r” indicates reverse primer. With reference to NC_003977. c An annealing temperature of 54°C was used during sequencing cycling to ensure a high rate of success of sequencing. b

due to low viral load in the plasma samples: (i) For primer sets 1a and 1b (251f-1190r and 595f-1797r), 3/126 were not amplifiable, with a median viral load of 31 IU/ml (range, 26 to 50 IU/ml); (ii) for primer set 2a (2300f-215r), 5/126 were not amplifiable, with a median viral load of 31 IU/ml (range, 26 to 137 IU/ml); (iii) for primer set 2b (2819f-617r), 5/126 were not amplifiable, with a mean viral load of 57 IU/ml (range, 26 to 137 IU/ml); (iv) for primer set 3 (1877f-2835r), 4/126 were not amplifiable, with a mean viral load of 36 IU/ml (range, 26 to 57 IU/ml); and (v) for primer set 4 (1584f-2331r), 4/126 were not amplifiable, with a mean viral load of 36 IU/ml (range, 26 to 58 IU/ml). The overall rate of success of sequencing for these samples was more than 94% for all primer sets, except primers 251f and 2300f, which had success rates of 92% and 89%, respectively (Table 4). From the 126 samples, genotypes B and C were the predominant genotypes, accounting for 72.0% (n ⫽ 90) and 25.6% (n ⫽ 32), respectively. Two samples had genotype D and one had genotype A. One sample was not amplifiable with all primer sets due to a very low viral load. Application of overlapping primer sets across HBV reference panel genotypes A to G. When the six primer sets were tested on 15 HBV strains from PEI, all gave 100% successful amplification and sequencing for all six primer sets. Primers 1190f and 2331r from sample 10 (genotype D) gave only a partial sequence. Using clonal sequencing, it was found that this was due to the presence of a deletion mutation near the 5= end upstream of the precore region. Primers 2331r and 1877f from sample 12 (genotype D) also

June 2015 Volume 53 Number 6

gave a partial sequence. Again, clonal sequencing was conducted, which revealed the cause to be the presence of a deletion mutation near the 3= end of the core region. As the “failures” with these primers were not purely due to direct sequencing failure, they were not included in the calculation of the rate of success of sequencing (Table 4). DISCUSSION

Based on 5,154 HBV genomes retrieved from the GenBank, six universal primer sets highly conserved across genotypes A to J were designed. These primer sets worked optimally, as they could achieve a very low limit of detection, 50 to 100 IU/ml. When applied to HBV genotypes A to D from Malaysian patients, high rates of success of amplification (96 to 98%) and sequencing (89 to 99%) were achieved for all primer sets. Similarly, the success rate was 100% for both amplification and sequencing in the evaluation of an HBV genotype panel provided by PEI. The ideal way to obtain a complete HBV haplotype genome is through a combined approach of restriction enzyme assay and clonal sequencing. However, this method requires suitable restriction sites and is often not applicable in samples of low viral load. There are two types of HBV genomes present in the blood of chronic hepatitis B patients: the majority is virion DNA, while the other, present in a rather lower copy number, is covalently closed circular DNA (cccDNA). A high failure rate has been reported for the use of a single primer set for the amplification of complete

Journal of Clinical Microbiology

jcm.asm.org

1833

Downloaded from http://jcm.asm.org/ on May 15, 2015 by Yale University

Typical amplicon size (bp)

Primer set and namesa

Chook et al.

TABLE 2 Annealing temperature of PCR and coverage for each primer set used for amplification of HBV genome fragments % Coverage in 5,154 HBV genomes retrieved from GenBank Ta (°C) of 2nd-round PCR

Set 1a 251f 1190r

54

Set 1b 595f 1797r 1190f

56

Set 2a 2300f 215r Set 2b 2819f 617r

50

Set 3 1859f 1877f 2835r

52

Set 4 1584f 2331r 2396r

57°C

500

100

50

50

50

NTC

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫺ ⫺ ⫺ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫺ ⫺ ⫹ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺

100% identity 98.22 98.43

99.71 99.38

1a 1b 2a 2b 3 4

98.88 97.32 98.12

99.48 98.80 99.40

a Amplification using 251f-1797r (outer primer set 1) followed by 251f-1190r (inner set 1a) and 595f-1797r (inner set 1b), 2300f-654r (outer primer set 2) followed by 2300f215r (inner set 2a) and 2819f-617r (inner set 2b), 1859f-2835r (outer primer set 3) followed by 1877f-2835r (inner set 3), and 1584f-2396r (outer primer set 4) followed by 1584f-2331r (inner set 4). NTC, no-template control.

94.55 97.77

99.19 99.57

96.49 98.78

98.66 99.75

97.75 97.75 96.70

98.99 99.50 99.32

97.87 96.97 97.94

99.44 99.05 99.44

50

Suffix “f” indicates forward primer, whereas “r” indicates reverse primer. The first 3 nucleotides are totally conserved near the 3= ends of all the primers to ensure a high rate of success of amplification.

b

genomes of HBV (12). This is because the primer set amplifies the cccDNA of HBV, instead of the virion DNA. Using a single primer set, the Taq polymerase cannot extend through the virion DNA due to the presence of breakpoints within the viral genome, one of which is located near nt 1600 in the positive strand and another near nt 1800 in the negative strand (13). Using one of the primer pairs in this study, outer set 1859f-1797r, followed by inner set 1877f-1797r, an effort to amplify a nearly complete HBV genome was made. Even though the primer-flanking region avoided the breakpoints, the lowest limit of detection that could be achieved was 500 IU/ml. This is about 5-fold less sensitive than the overlapping primer sets. In addition, the nearly complete genome primers had a higher chance of nonspecific amplification of smaller fragments than did the overlapping primer sets. Although the nearly complete genome primer sets may help to save some PCR reagents in the first round of amplification, the use of the later primer sets seems to be a more practical approach, as it could amplify samples with a wide range of viral loads, especially those with very low viral loads. Designing universally conserved primer sets across the complete genome of the virus remains challenging due to the errorprone reverse transcription strategy in HBV replication. The median coverages for primer sets described in previous studies were 88% (range, 87 to 89%) (7), 90% (range, 1 to 98%) (14), and 74% (range, 27 to 89%) (15). Considering the high DNA sequence

jcm.asm.org

Primer set

Allowance of one nucleotide mismatchb

a

1834

Positivity (IU/ml)a

divergence in HBV genomes, it is not surprising that some of the previously described primer sets may fail to amplify when applied to samples with different genotypes or from different geographical origins. In this study, the median coverage was 98% (95 to 99%), which was higher than in the aforementioned studies. This was probably one of the major factors contributing to the high amplification rate in the PCR assays for all primer sets, ⬎96%, in this study. Finding a primer set that works well in both PCR amplification and sequencing adds to the complexity of the design. To ensure efficient amplification, a primer set that is highly prone to the formation of secondary structure and primer-dimers would be eliminated from further evaluation. In this study, all primer sets chosen had a limit of detection as low as 100 IU/ml; some primer sets reached a much lower limit, 50 IU/ml, albeit inconsistently. The 50-IU/ml level, when adjusted to the starting extracting plasma volume of 1 ml, corresponds to 10 IU/ml. This value is comparable to the limit of quantification in the Roche Cobas TaqMan HBV DNA quantification assay (ⱖ20 IU/ml). The standard of 50 IU/ml was run in triplicate. It was found that not all of the 50-IU/ml standards were amplifiable, probably due to the effect of Poisson distribution, where some levels may be too low to allow the detection of a single copy of virus each time. To reduce the risk of sequencing failure, all primer sets were checked for 8-mer redundancy within the amplified regions of 10 HBV genomes of genotypes A to J during the primer design (results not shown). Based on 126 Malaysian isolates (mainly genotypes B and C) and 15 PEI samples (genotypes A to G), all primer sets generally worked well, having a rate of successful sequencing of 92% or above, except for primer 2300f. The reason for the slightly lower rate of success of sequencing for primer 2300f is probably the higher sequence divergence in the primer binding region, which could be reflected from a rather lower coverage of this primer (Table 2). Other primer sets designed near the primer 2300f region, such as nt 2261 to 2277, nt 2306 to 2325, and nt 2368 to 2388, have been tried. All resulted in either nonspecific amplification or sequencing failure. In conclusion, universal overlapping primer sets covering HBV complete genomes have been established in this study. They could reliably amplify and sequence HBV complete genomes of genotypes A to G with high success.

Journal of Clinical Microbiology

June 2015 Volume 53 Number 6

Downloaded from http://jcm.asm.org/ on May 15, 2015 by Yale University

Primer set and namesa

TABLE 3 Limit of detection for 6 primer sets used in the amplification of HBV genomes

Universal Primers for Sequencing of HBV Genomes

TABLE 4 Success rates of six primer sets in amplification and sequencing of HBV genomes Malaysian isolates n ⫽ 126 No. (%) with successful PCR amplification

Set 1a 251f 1190r

123 (97.6)

Set 1b 595f 1190f

122 (96.8)

Set 2a 2300f 215r

121 (96.0)

Set 2b 2819f 617r

121 (96.0)

Set 3 1877f 2835r

122 (96.8)

Set 4 2331r

122 (96.8)

No./total (%) with successful direct sequencing

No. (%) with successful PCR amplification

No./total (%) with successful direct sequencing

113/123 (91.9) 120/123 (97.6)

15 (100.0) 15 (100.0)

15/15 (100.0) 15/15 (100.0)

121/122 (99.2) 120/122 (98.4)

15 (100.0) 15 (100.0)

15/15 (100.0) 14/14 (100.0)a

108/121 (89.3) 116/121 (95.9)

15 (100.0) 15 (100.0)

15/15 (100.0) 15/15 (100.0)

119/121 (98.3) 114/121 (94.2)

15 (100.0) 15 (100.0)

15/15 (100.0) 15/15 (100.0)

121/122 (99.2) 116/122 (95.1)

15 (100.0) 15 (100.0)

14/14 (100.0)b 15/15 (100.0)

120/123 (98.4)

15 (100.0)

13/13 (100.0)a,b

a

Sample 10 (genotype D) from Paul-Erhlich-Institut (PEI code 5086/08) showed some background noises in primers 1190f and 2331r. Further investigation found that they were not a pure sequencing failure. Clonal sequencing (20 clones) discovered that it contained a mixed infection of a wild type and multiple deletion mutants. Hence, they were not included in the calculation for the success rate in direct sequencing. b Sample 12 (genotype D) from Paul-Erhlich-Institut (PEI code 5086/08) showed high background noises in primers 1877f and 2331r. Further investigation found that they were not a pure sequencing failure. Clonal sequencing (20 clones) discovered that it contained a mixed infection of a wild type and multiple deletion mutants. Hence, they were not included in the calculation for the success rate in direct sequencing.

ACKNOWLEDGMENTS This study was sponsored by a Universiti Malaya High Impact Research (HIR) Grant (UM.C/625/1/HIR/MOHE/MED/25@H-20001-E0063) from the Ministry of Higher Education (MOHE). We thank all the UM and UMMC staff that helped in this study. We also thank Micha Nübling and others from Paul-Ehrlich-Institut (PEI) for the generous gift of the First WHO Reference Panel for Hepatitis B Virus Genotypes.

REFERENCES

9.

10.

1. Beck J, Nassal M. 2007. Hepatitis B virus replication. World J Gastroenterol 13:48 – 64. http://dx.doi.org/10.3748/wjg.v13.i1.48. 2. Harrison A, Lemey P, Hurles M, Moyes C, Horn S, Pryor J, Malani J, Supuri M, Masta A, Teriboriki B, Toatu T, Penny D, Rambaut A, Shapiro B. 2011. Genomic analysis of hepatitis B virus reveals antigen state and genotype as sources of evolutionary rate variation. Viruses 3:83– 101. http://dx.doi.org/10.3390/v3020083. 3. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Imai M, Miyakawa Y, Mayumi M. 1988. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 69:2575–2583. http://dx.doi.org/10.1099/0022-1317-69-10-2575. 4. Ngui SL, Teo CG. 1997. Hepatitis B virus genomic heterogeneity: variation between quasispecies may confound molecular epidemiological analyses of transmission incidents. J Viral Hepat 4:309 –315. http://dx.doi.org /10.1046/j.1365-2893.1997.00066.x. 5. Yim HJ, Hussain M, Liu Y, Wong SN, Fung SK, Lok AS. 2006. Evolution of multi-drug resistant hepatitis B virus during sequential therapy. Hepatology 44:703–712. http://dx.doi.org/10.1002/hep.21290. 6. Enomoto M, Tamori A, Kohmoto MT, Morikawa H, Habu D, Sakaguchi H, Takeda T, Seki S, Kawada N, Shiomi S, Nishiguchi S. 2007. Mutational patterns of hepatitis B virus genome and clinical outcomes after emergence of drug-resistant variants during lamivudine therapy: analyses of the polymerase gene and full-length sequences. J Med Virol 79:1664 –1670. http://dx.doi.org/10.1002/jmv.20984. 7. Li W, Chen G, Yu X, Shi Y, Peng M, Wei J. 2013. Accumulation of the

June 2015 Volume 53 Number 6

8.

11.

12.

13.

14.

15.

mutations in basal core promoter of hepatitis B virus subgenotype C1 increase the risk of hepatocellular carcinoma in Southern China. Int J Clin Exp Pathol 6:1076 –1085. Osiowy C, Giles E, Tanaka Y, Mizokami M, Minuk GY. 2006. Molecular evolution of hepatitis B virus over 25 years. J Virol 80:10307–10314. http: //dx.doi.org/10.1128/JVI.00996-06. Shi W, Carr MJ, Dunford L, Zhu C, Hall WW, Higgins DG. 2012. Identification of novel inter-genotypic recombinants of human hepatitis B viruses by large-scale phylogenetic analysis. Virology 427:51–59. http: //dx.doi.org/10.1016/j.virol.2012.01.030. Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059 –3066. http://dx.doi.org/10.1093/nar/gkf436. Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98. Günther S, Sommer G, Von Breunig F, Iwanska A, Kalinina T, Sterneck M, Will H. 1998. Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations. J Clin Microbiol 36:531–538. He ML, Wu J, Chen Y, Lin MC, Lau GK, Kung HF. 2002. A new and sensitive method for the quantification of HBV cccDNA by real-time PCR. Biochem Biophys Res Commun 295:1102–1107. http://dx.doi.org/10 .1016/S0006-291X(02)00813-6. Xu Z, Liu Y, Xu T, Chen L, Si L, Wang Y, Ren X, Zhong Y, Zhao J, Xu D. 2010. Acute hepatitis B infection associated with drug-resistant hepatitis B virus. J Clin Virol 48:270 –274. http://dx.doi.org/10.1016/j.jcv.2010 .05.010. Sa-nguanmoo, P, Thongmee Ratanakorn C, Pattanarangsan P, Boonyarittichaikij R, Chodapisitkul R, Theamboonlers S, Tangkijvanich A, Poovorawan PY. 2008. Prevalence, whole genome characterization and phylogenetic analysis of hepatitis B virus in captive orangutan and gibbon. J Med Primatol 37:277–289. http://dx.doi.org/10.1111/j.1600 -0684.2008.00290.x.

Journal of Clinical Microbiology

jcm.asm.org

1835

Downloaded from http://jcm.asm.org/ on May 15, 2015 by Yale University

Primer set and names

Paul-Erhlich-Institut isolates, n ⫽ 15

Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G.

Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional ...
314KB Sizes 1 Downloads 12 Views