Wangdon Yoo

Increased risk of adefovir resistance in patients with lamivudine‐resistant chronic hepatitis B after 48 weeks of adefovir dipivoxil monotherapy

Although adefovir dipivoxil (ADV) has a unique profile of delayed and infrequent resistance in treatment‐naïve chronic hepatitis B patients, the association of ADV resistance with previous lamivudine (LAM) resistance is not well understood. We compared the emergence of the ADV‐resistant mutations rtA181V/T and rtN236T between LAM‐resistant patients and treatment‐naïve patients at 48 weeks of ADV monotherapy. Fifty‐seven LAM‐resistant patients and 38 treatment‐naïve patients were treated with 10 mg/d ADV for more than 48 weeks. Both baseline and 48‐week blood samples were analyzed for ADV‐resistant mutations via restriction fragment mass polymorphism analysis. Antiviral responses were evaluated according to changes in serum HBV DNA (measured via real‐time polymerase chain reaction) and alanine aminotransferase (ALT) levels and loss of hepatitis B e antigen (HBeAg). After 48 weeks, 10 (18%) of the 57 LAM‐resistant patients were found to have developed ADV‐resistant mutations, whereas none of the 38 treatment‐naïve patients developed such mutations (P < .01). Among LAM‐resistant patients, the reduction in serum HBV DNA levels was significantly lower in patients with ADV‐resistant mutations than in those without such mutations (−1.04 vs. −2.63 log10 copies/mL) (P = .01). However, the rates of serum ALT normalization (60% vs. 55%) and HBeAg loss (14% vs. 21%) were not significantly different between the 2 groups (P > .05). In conclusion, the emergence of the rtA181V/T and rtN236T mutations was more common in LAM‐resistant patients than in treatment‐naïve patients after 48 weeks of ADV therapy and was associated with reduced antiviral efficacy to drug treatment. (HEPATOLOGY 2006;43:1385–1391.)

Resistance to adefovir dipivoxil in lamivudine resistant chronic hepatitis B patients treated with adefovir dipivoxil

Background: Adefovir dipivoxil (ADV) is a potent nucleotide analogue against both the wild-type and lamivudine (LMV) resistant hepatitis B virus (HBV). The cumulative incidence of ADV resistant mutations in the nucleoside/-tide treatment naïve chronic hepatitis B patient (CHB) at weeks 48, 96, and 144 was 0, 0.8–3%, and ∼5.9%, respectively. Aims: The aim of this study was to characterise the genotypic and phenotypic mutation profiles to ADV in 67 LMV resistant CHB patients who were treated with ADV. Methods: Serum HBV DNA was quantified by real time polymerase chain reaction. The ADV mutant was detected using matrix assisted laser desorption/ionisation time of flight mass spectrometry based genotyping assays, termed restriction fragment mass polymorphism (RFMP). Results: RFMP analysis revealed that a total of 11 amino acid substitutions developed in the rt domain of the HBV polymerase in nine patients. The cumulative incidence of genotypic ADV resistance at months 12 and 24 was 6.4% and 25.4%, respectively. The rtA181V, rtN236T, and rtA181T mutations were detected in five, four, and two of the 67 patients at treatment months 12–17, 3–19, and 7–20, respectively. Serial quantification of serum HBV DNA revealed that two patients with the rtA181V mutation, with or without the rtN236T mutation, and one patient with the rtA181T mutation displayed HBV DNA rebound. Conclusion: Emergence of the ADV mutation in LMV resistant patients who are treated with ADV appeared to present earlier and more frequently than was reported in previous studies on nucleoside/-tide treatment naïve patients.

A purified inactivated Japanese encephalitis virus vaccine made in vero cells

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

High-resolution human papillomavirus genotyping by MALDI-TOF mass spectrometry

We describe a matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) mass spectrometry (MS)-based assay for human papillomavirus (HPV) genotyping—the restriction fragment mass polymorphism (RFMP) assay, which is based on mass measurement of genotype-specific oligonucleotide fragments generated by TypeIIS restriction endonuclease cleavage after recognition sites have been introduced by PCR amplification. The use of a TypeIIS restriction enzyme makes the RFMP assay independent of sequence and applicable to a wide variety of HPV genotypes, because these enzymes have cleavage sites at a fixed distance from their recognition sites. After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS. Overall, the protocol is simple, takes ∼4–4.5 h and can accurately detect and identify at least 74 different HPV genotypes.

Mutational complex genotype of the hepatitis B virus X /precore regions as a novel predictive marker for hepatocellular carcinoma

This study explored the combined effect of number and pattern of mutations in the X/precore regions of the hepatitis B virus (HBV) genome, mutational complex genotype (MCG), on hepatocellular carcinoma (HCC) development. Sequence variations were determined by direct sequencing and multiplex restriction fragment mass polymorphism analysis in 150 age‐, sex‐ and hepatitis B e antigen (HBeAg) status‐matched patients with and without HCC. In addition, a longitudinal study and an external validation of MCG were conducted. All were HBV subgenotype C2. Eight high‐frequency mutations (G1613A, C1653T, T1753V, A1762T, G1764A, A1846T, G1896A and G1899A) were significantly associated with HCC. Whereas C1653T, T1753V, G1764A and A1846T were independent mutational factors for HCC, the significance of these individual mutations was negligible when analyzed with all clinico‐virological variables. The total number of mutations was the only independent viral factor for HCC, irrespective of HBeAg status. There was a significant dose–risk relationship between the number of mutations and HCC, in which high risks for HCC were associated with mutation numbers ≥6. Pattern analysis of the mutations revealed disparity in distribution among the top seven high‐risk mutation combination patterns, which accounted for 40 and 2.7% of HCC and non‐HCC cases, respectively. The predictive accuracy of the high‐risk mutations for HCC was similar to that of α‐fetoprotein. Longitudinal and external validation studies also supported the association of mutation number with HCC development. MCG in the HBV X/precore regions is a risk indicator for HCC, and might serve as a new guide to the HCC screening scheme for chronic HBV carriers. (Cancer Sci 2012; 103: 296–304)

Antiviral therapies: focus on Hepatitis B reverse transcriptase

Hepatitis B virus (HBV) is the etiologic agent of mankinds most serious liver disease. While the availability of a vaccine has reduced the number of new HBV infections, the vaccine does not benefit the approximately 350 million people already chronically infected by the virus. Most of the drugs approved by the FDA for the treatment of hepatitis B target the reverse transcriptase (RT or P gene product) and are nucleoside RT inhibitors (NRTIs) that suppress viral replication. However, prolonged monotherapies directed against a single target result in the emergence of viral resistance. HBV genotypic differences affect NRTI resistance, and because the reading frames of the S (surface antigen) and P genes partially overlap, genomic differences that affect the surface of the virus may also alter the viral polymerase sequence, function and drug susceptibility. The scope of this review is to assess the effects of HBV genotypic variation on the development of drug resistance to NRTIs. Some RT residues that vary among different genotypes are in the vicinity of residues that mutate and give rise to NRTI resistance. Interactions between these amino acids can help explain the effect of HBV genotype on the development of NRTI resistance during antiviral therapies, and might help in the design of improved therapeutic strategies.

Levels of Hepatitis B Virus (HBV) Replication During the Nonreplicative Phase: HBV Quantification by Real-Time PCR in Korea

The levels of HBV replication in the nonreplicative phase are not clear. We conducted this study to evaluate the levels of viral replication during the nonreplicative phase in chronic HBV-infected Korean patients using real-time PCR. A total of 125 patients were classified into three groups: inactive HBsAg carriers, inactive liver cirrhosis patients, and resolved chronic HBV-infected patients with loss of HBsAg. The real-time PCR detected HBV DNA in 112 cases (89.6%). The mean levels of HBV DNA were 3.84, 4.10, and 3.31 log copies/ml in the three groups, respectively (P <0.01). Ninety-five percent of inactive HBsAg carriers showed levels of HBV DNA lower than 6×104 copies/ml. In conclusion, we showed different levels of HBV DNA exactly in three groups during nonreplicative phases. We suggest that the cutoff level of HBV DNA in inactive HBsAg carriers should be readjusted to a lower level in future studies.

Nucleotide Sequence of Envelope Protein of Japanese Encephalitis Virus SA14-14-2 Adapted to Vero Cells

Live attenuated Japanese encephalitis (JE) virus SA14-14-2 produced in primary dog kidney cells (PDK) was adapted to Vero cells. In an effort to gain insight into the molecular basis of the biological characteristics of the SA14-14-2(Vero) strain, the 1500 nucleotide sequence encoding the envelope (E) gene which possesses major neutralizing epitopes was determined and compared with the sequences of two other attenuated JE virus strains, SA14-14-2(PHK) and SA14-14-2(PDK). The amino acid sequence of the C-terminal region (a.a. 280-500) was found to be identical for all three strains, while the N-terminal region (a.a. 1-279) shows sequence variation. The distribution of mutations in the N-terminal region was nearly the same among the three attenuated strains, suggesting that the N-terminal sequences might be related with virus-host cell specificity. However, it was found that Lys and Val (a.a. 138 and 176, respectively), known to be responsible for attenuation, are still conserved in SA14-14-2(Vero). Animal testing showed that SA14-14-2(Vero) has an attenuation phenotype similar to that of the parent s SA14-14-2(PDK) strain in mice.

A simple and accurate SNP scoring strategy based on typeIIS restriction endonuclease cleavage and matrix-assisted laser desorption/ionization mass spectrometry

BackgroundWe describe the development of a novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-based single nucleotide polymorphism (SNP) scoring strategy, termed Restriction Fragment Mass Polymorphism (RFMP) that is suitable for genotyping variations in a simple, accurate, and high-throughput manner. The assay is based on polymerase chain reaction (PCR) amplification and mass measurement of oligonucleotides containing a polymorphic base, to which a typeIIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products leads to excision of oligonucleotide fragments representing base variation of the polymorphic site whose masses were determined by MALDI-TOF MS.ResultsThe assay represents an improvement over previous methods because it relies on the direct mass determination of PCR products rather than on an indirect analysis, where a base-extended or fluorescent report tag is interpreted. The RFMP strategy is simple and straightforward, requiring one restriction digestion reaction following target amplification in a single vessel. With this technology, genotypes are generated with a high call rate (99.6%) and high accuracy (99.8%) as determined by independent sequencing.ConclusionThe simplicity, accuracy and amenability to high-throughput screening analysis should make the RFMP assay suitable for large-scale genotype association study as well as clinical genotyping in laboratories.

Preparation of a purified, inactivated Japanese encephalitis (JE) virus vaccine in Vero cells

An attenuated Japanese encephalitis (JE) virus SA14-14-2 (PDK) was adapted to Vero cells, a continuous cell line that has been licensed for human vaccine production, by serial passages. The resulting virus was purified by tangential flow ultrafiltration followed by sucrose density gradient ultracentrifugation, giving 2.3 mg purified virus per liter of culture supernatant. Treatment with 0.05% formalin for 4 days at 22 °C completely inactivated viral infectivity while preserving its antigenicity. The purified, inactivated JE virus was formulated with alum hydroxide and administered to mice by intraperitoneal route. In terms of its ability to induce anti-JE neutralizing antibody and to protect the immunized animal against neurovirulent virus challenge, the purified, inactivated JE virus formulated with alum was equivalent to the exiting commercial mouse brain-derived vaccine (JE-VAX, Aventis Pasteur Inc.).