Identification of rare polymerase variants of hepatitis B virus using a two‐stage PCR with peptide nucleic acid clamping

Ohishi, Waka; Shirakawa, Hiroo; Kawakami, Yoshiiku; Kimura, Shinzo; Kamiyasu, Masaya; Tazuma, Susumu; Nakanishi, Toshio; Chayama, Kazuaki

doi:10.1002/jmv.20026

Cited by 33 publications

(53 citation statements)

References 38 publications

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“…Existence of HBV quasi-species (Schatzl et al, 1997) has facilitated the development of mutants with specific ability to escape antibody detection and neutralization. These mutants may lead to reinfection, because it replicates through an RNA intermediate synthesized by reverse transcriptase of viral genomes (Kreutz, 2002;Ohishi et al, 2004) and quasi-species are generated (Torresi, 2002;Liu et al, 2002;Hsu et al, 2004). This resulted in the production of viral mutants during naturally occurring infections (Chong-Jin et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Identification of Novel D11 Hepatitis B Surface Antigen Subgenotype in Jeddah, Kingdom of Saudi Arabia

Hadad¹,

Alakilli²,

Rabah³

et al. 2015

Biotechnology

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A B S T R A C T Hepatitis B virus (HBV) infection remains to be a worldwide health problem. In Saudi Arabia, HBV is the most predominant type of hepatitis followed by hepatitis C and hepatitis A while little is known about the molecular epidemiology of the prevalence of HBV genotype/subgenotype particularly in Jeddah. Serum samples were collected from HBV chronic patients and subjected to HBsAg gene amplification. Sequencing and phylogenetic analysis of the entire HBsAg gene sequences revealed that 11 isolates belonged to HBV/D while 4 isolates were associated with HBV/C. Interestingly, HBV/D subgenotypes identified eight HBV/D present isolates belonged to HBV/D1 while three isolates showed a new cluster supporting by a branch with 99% bootstrap value and 4.3-5.8% nucleotide divergence over the entire HBsAg gene from other known subgenotypes D1 to D10, despite they were appearing more related to HBV/D5. The three strains of the new D subgenotype showed unique amino acid sequences consisting of Thr7non, 75Pro in the preS1 gene, 112Ile, 161Gly in the preS2 gene and 196Glu, 197Ala, 238Ser, 259Cys in the S gene. In addition to three amino acid residues in the S gene (373Ile, 374Ala and 381Thr) were specified S118S isolate. Subsequently, it have been verified that HBV/D1 is the most prevalent HBV subgenotype in Jeddah as well as we proposed a novel subgenotype designated HBV/D11. The identification of HBV/D11 novel subgenotypes in the present study suggested that further studies with a large number of subjects in previously examined and unexamined areas may lead to discovering new HBV strain genotypes and/or subgenotypes circulating in Saudi Arabia.

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Section: Discussionmentioning

confidence: 99%

Identification of Novel D11 Hepatitis B Surface Antigen Subgenotype in Jeddah, Kingdom of Saudi Arabia

Hadad¹,

Alakilli²,

Rabah³

et al. 2015

Biotechnology

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“…In principle, this does not aberrantly alter the wild-type sequences and leads to much lower false-positive rates. 3 The PNA/LNA-mediated clamping technique has been used for over a decade and is an attractive method that has proven to be potentially applicable to many situations, including the examination of various cancer mutations, 8,19 -23 identification of viral or bacterial variants, 24,25 genotyping for single-base substitutions, 26,27 detection of low-level somatic mosaicism 28 or mitochondrial heteroplasmy, 29,30 and prenatal diagnosis via maternal plasma, 31 among many others. However, the high cost and limited availability have restricted its usage.…”

Section: Discussionmentioning

confidence: 99%

Mutant Enrichment with 3′-Modified Oligonucleotides

Lee

Kim

Kown

et al. 2011

The Journal of Molecular Diagnostics

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Many clinical situations necessitate highly sensitive and reliable molecular assays; however, the achievement of such assays remains a challenge due to the inherent limitations of molecular testing methods. Here, we describe a simple and inexpensive enrichment technique that we call mutant enrichment with 3=-modified oligonucleotides (MEMO). The method is based on the use of a 3=-modified oligonucleotide primer that blocks extension of the normal allele but enables extension of the mutated allele. The performance of the technique was evaluated with respect to its ability to detect common cancer mutations in the EGFR, KRAS, BRAF, TP53, JAK2, and NPM1 genes. We achieved sensitivities of 10 ؊2 to 10 ؊6 using downstream Sanger sequencing, depending on the concentrations and thermodynamics of the primers. MEMO

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“…PNAs are DNA mimics in which the deoxyribose phosphate backbone of DNA has been replaced by N-(2-aminoethyl)glycine linkages (30). They recognize and bind to their complementary nucleic acid sequences with high thermal stability and specificity (31).…”

Section: Inhibition Of Disease Progression For Patients With Chronicmentioning

confidence: 99%

“…PNAs are DNA mimics that bind to their complementary nucleic acid sequences with high specificity and have PCRclamping effects (30,31). We evaluated the efficacy of this assay and demonstrated the clinical significance of estimating the quantitative follow-up of dual mutants.…”

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confidence: 99%

Sensitive Assay for Quantification of Hepatitis B Virus Mutants by Use of a Minor Groove Binder Probe and Peptide Nucleic Acids

et al. 2010

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Lamivudine is the first nucleoside analogue that was shown to have a potent effect on hepatitis B virus (HBV). However, the emergence of mutants resistant or cross-resistant to nucleoside/nucleotide analogues remains a serious problem. Several assays for the detection and quantification of antiviral-resistant mutants have been reported, but it has been difficult to measure the amounts of mutants accurately, especially when the target strain is a minor component of the mixed population. It has been shown that accurate measurement of a minor strain is difficult as long as a matching reaction with a single probe is included in the assay. We developed a new method for the quantification of lamivudine-resistant strains in a mixed-virus population by real-time PCR using minor groove binder probes and peptide nucleic acids, and we achieved a wide and measurable range, from 3 to 10 log 10 copies/ml, and high sensitivity, with a discriminative limit of 0.01% of the predominant strain. The clinical significance of measuring substitutions not only of M204 but also of L180 residues of HBV polymerase was demonstrated by this method. This assay increases the versatility of a sensitive method for the quantification of a single-nucleotide mutation in a heterogeneous population.Chronic liver diseases due to hepatitis B virus (HBV) infection are still serious problems worldwide, and many patients suffer from liver cirrhosis and hepatocellular carcinoma (2, 6, 21). Nucleoside/nucleotide analogues have been introduced in the treatment of chronic hepatitis B, and inhibition of disease progression has been achieved (20). However, since several analogues have been developed and have come into wide use, the risk of the emergence of resistant or cross-resistant mutations is increasing. This is a serious problem, since it may lead to the virological and biochemical breakthroughs that result in the loss of therapeutic effects (18).Lamivudine (3TC) is the first nucleoside analogue that was demonstrated to have a potent effect on HBV (8, 17). The results of many studies on lamivudine-resistant mutants have been reported, and the impact of alteration from M204 to I204 or V204 (YMDD mutations) was initially investigated (20). Thereafter, the clinical significance of other regions of HBV polymerase was clarified. We reported the significance of dual mutations of the YMDD motif and the LLAQ motif (specifically the L180 residue) for the severity of breakthrough hepatitis (29). Recently, alteration of the L180 residue has been noted as one of the most crucial changes for the emergence of entecavir-resistant mutants.It is important to detect mutants as early as possible in order to confirm genotypic or phenotypic resistance that may be followed by virological breakthrough and rebound. Several methods using PCR or hybridization for the qualitative detection of antiviral-resistant mutants have been reported. Some assays have been proposed as sensitive methods for the quantitative measurement of mutant strains (33), and we have reported a quantificat...

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Identification of rare polymerase variants of hepatitis B virus using a two‐stage PCR with peptide nucleic acid clamping

Cited by 33 publications

References 38 publications

Identification of Novel D11 Hepatitis B Surface Antigen Subgenotype in Jeddah, Kingdom of Saudi Arabia

Identification of Novel D11 Hepatitis B Surface Antigen Subgenotype in Jeddah, Kingdom of Saudi Arabia

Mutant Enrichment with 3′-Modified Oligonucleotides

Sensitive Assay for Quantification of Hepatitis B Virus Mutants by Use of a Minor Groove Binder Probe and Peptide Nucleic Acids

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