Cross-resistance profile determination of two second-generation HIV-1 integrase inhibitors using a panel of recombinant viruses derived from raltegravir-treated clinical isolates.

The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG.

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain. The ERASE-2 Study Group.

OBJECTIVE: To examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. METHODS: A cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively. RESULTS: Primary resistance to NRTI was recognized in nine out of 52 (17%) naive individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P< 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized. CONCLUSION: The prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

Prevalence of genotypic resistance among antiretroviral drug-naive HIV-1-infected patients in Belgium.

OBJECTIVES: To estimate the prevalence and the evolution over time (1995-1998) of genotypic resistance to antiviral drugs in antiretroviral drug-naive HIV-1-infected patients in Belgium. DESIGN: Belgian Aids Reference Laboratories provided retrospective samples and clinical data from antiretroviral drug-naive HIV-1-infected patients who visited the hospital for the first time in 1995 (n=45), 1997 (n=75) and 1998 (n=111). Genotypic resistance to the three available classes of drugs was monitored using the Line Probe Assay (Innogenetics, Gent, Belgium). Additionally, ARMS-151 was performed for scoring multinucleoside resistance. RESULTS: The prevalence of genotypic resistance at baseline to nucleoside analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were each between 10% and 20% for 1995, 1997 and 1998 without an increasing trend over time. For NRTIs, resistance mutations were mainly related to zidovudine in 1995, whereas in 1997 and 1998 baseline resistance was scored for zidovudine, lamivudine or for both drugs simultaneously. No patients displayed the multi-nucleoside resistance Q151M mutation. Baseline resistance mutations to protease inhibitors (PIs) did not rise significantly: 4.4% in 1995, 8% in 1997 and 9.9% in 1998. When scoring any resistance-related mutation, 26.6% displayed genotypic baseline resistance in 1995, 26.6% in 1997 and 31.5% in 1998. DISCUSSION: The prevalence of genotypic baseline resistance to any drug, as scored with LiPA, in naive HIV-1 patients in Belgium is 29%, with baseline resistance mutations to one or several drugs from all available classes of antiviral drugs. The ability of LiPA to pick up minor variants could be an explanation for the higher overall prevalence we observe, when compared to recent estimates in other countries of 16.3% and 22%, which were based on sequencing methods. According to the European guidelines for resistance testing, resistance testing in Belgium before starting antiviral therapy should be considered.

Hepatitis C virus genotyping by means of 5'-UR/core line probe assays and molecular analysis of untypeable samples.

To test the theoretical possibility of 5'-UR mistyping between hepatitis C virus subtypes 1a and 1b, we combined a 5'-UR/Core line probe assay (LiPA) with a nested PCR system and retested 183 sera, previously genotyped as type 1a or 1b and originating mainly from Western Europe. Eight percent of these were found to be wrongly subtyped. Based on this method, 3 additional subtypes of type 1 were discovered (1d-1f). Randomly selected European type 2 sera (n = 18) were tested with a similar type 2 5'-UR/Core LiPA. They were unexpectedly found to belong to subtype 2c in the majority of cases. Among serum samples originating from South-East Asia, several additional genotypes (7a, 7c, 7d, and 9a) were detected which had 5'-UR sequence motifs indistinguishable from genotype 1. Based on 13,203 pairwise comparisons in the 340-bp NS5B region, classification into types, subtypes, and isolates was obtained in 99.8% of all cases by using the phylogenetic border value of 0.328 for subtypes/types and 0.127 for isolates/subtypes; and evidence for a 10th major type of HCV was provided. Combination of all available HCV sequence data from the 447-bp Core/E1 region and the NS5B 340-bp and 222-bp regions provided evidence for the existence of 10 types, including 50 subtypes. Previously, extensive studies involving genotypes 1a, 1b, 2a, and 2b indicated the importance of HCV subtyping in interferon treatment and progression of chronic liver disease. The herein described expansion in the number of HCV types and subtypes should help improve diagnosis, treatment and possibly prophylaxis of hepatitis C liver disease.

Baseline HIV type 1 genotypic resistance to a newly added nucleoside analog is predictive of virologic failure of the new therapy.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

Comparison of the LiPA HIV-1 RT test, selective PCR and direct solid phase sequencing for the detection of HIV-1 drug resistance mutations.

The performance to detect drug resistance mutations in the reverse transcriptase gene of HIV-1 was compared for direct solid phase sequencing, selective polymerase chain reaction (PCR) using the amplification refractory mutation system (ARMS) and the new line probe assay (LIPA) HIV-1 RT. The three tests were undertaken on 50 plasma samples from 25 treatment-experienced patients under combination therapy with dideoxynucleoside analogues. LiPA HIV-1 RT gave interpretable results in 80 to 96% of the samples depending on the codon of interest. In 2% of the samples a failure to amplify resulted in uninterpretable results for sequencing. ARMS gave no result in seven samples (14%). Overall, there was a 73 to 100% concordance between the three methods. In this study, LiPA HIV-1 RT proved to be an accurate and reliable alternative to DNA sequencing for the detection of drug resistance mutations in patient samples.

Early detection of mixed mutations selected by antiretroviral agents in HIV-infected primary human lymphocytes.

A growing concern in the pursuit of new therapies for HIV-1 infection is the potential for the virus to develop drug resistance. With the advent of modern antiretroviral therapy and the common use of combined modalities, it is difficult to identify in the clinic the mutations associated with a specific drug. In general, drug selection of mutants using a relevant cell system, such as primary human lymphocytes, is a good prognosticator of what will happen in humans. In this study, HIV-infected human peripheral blood mononuclear cells were exposed, at a concentration of 1- to 10-fold the median effective antiviral concentration, to the nucleosides (-)-beta-2',3'-dideoxy-3'-thia-5-fluorocytidine [(-)-FTC] (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC), 3'-azido-2',3'-dideoxyuridine (CS-87, AZDU), 3'-azido-2',3'-dideoxy-5-methylcytidine (CS-92, AZMC), 2',3'-didehydro-3'-deoxythymidine (d4T), beta-L-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (beta-L-D4FC), beta-L-2',3'-dideoxyadenine SATE[beta-L-ddAMP-bis(tbutylSATE)], beta-L-5-fluoro-2',3'-dideoxycytidine (L-FddC), and the protease inhibitors nelfinavir and amprenavir (VX-478). Virus from the culture supernatant was amplified by PCR and analysed by both HIV-1 reverse transcriptase and protease line probe assay. All the L-nucleoside analogues tested selected for the V184 mutation, including the L-pyrimidine nucleosides 3TC (-)-FTC, beta- L-FddC, beta-L-D4FC and the beta-L-purine nucleoside. beta-L-D4FC also selected for K/R65 in addition to V184, indicating that these two mutations are linked and compatible in vitro. No pattern of mutations leading to resistance or reduced susceptibility was discerned with d4T. Rapid genotyping analysis revealed the different kinetics and mutations obtained by in vitro selection in HIV-infected cells exposed to nucleoside analogues and protease inhibitors.

Host and viral characteristics affecting the response to interferon therapy in chronic hepatitis C.

OBJECTIVE: To define parameters determined before and after 4 weeks of interferon therapy (3 MU three times per week for 24 weeks) which could be reliable predictors of a response to therapy. PATIENTS: Thirty-four patients with chronic hepatitis C virus (HCV) infection were investigated prospectively. METHODS: A complete response was defined as the normalization of serum alanine aminotransferase levels (ALT) at the end of treatment. The genotype of HCV was determined and the level of HCV-RNA was quantitated both before and after 4 weeks of treatment. RESULTS: After 4 weeks, 16 out of 20 responders [95% confidence interval (CI) 54-94%] and two out of 14 non-responders (95% CI 2-44%) normalized their ALT levels (P = 0.0002). The prevalence of genotype 1b was significantly (P < 0.04) higher among non-responders (eight out of 10; 95% CI 44-92%) than in responders (four out of 18; 95% CI 4-40%). Before treatment, the viraemia determined by branched DNA was significantly lower in responders than in non-responders (46.4 versus 116 x 10(5) eq virus/ml). After 4 weeks of treatment, the level of viraemia in responders was still significantly lower than that in non-responders (22.8 versus 66 x 10(5) eq virus/ml). In responders, a significant decrease in the level of viraemia was observed after 4 weeks of treatment. CONCLUSION: In a stepwise regression analysis only age and the normalization of ALT levels after 4 weeks of treatment were predictive of response to interferon at the end of treatment.

HCV Genotyping by the Line Probe Assay INNO-LiPA HCV II.

Hepatitis C viruses (HCVs) constitute a highly variable genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes a polyprotein which is co- and posttranslationally cleaved into at least nine proteins. Core, E1, and E2 (the structural proteins) and NS2, NS3, NS4A, NS4B, NS5A, and NS5B (the nonstructural [NS] proteins). A theoretical protein of 7 kDa (tp7) may be processed from the carboxy terminal E2 region (1).

Line Probe Assay for Detecting Mutations in HIV-1 Reverse Transcriptase.

The human immunodeficiency virus type 1 (HIV-1) belongs to the family of positive-stranded, enveloped RNA viruses with a DNA intermediate step (retroviruses). Because of the lack of fidelity of the reverse transcriptase (RT), the replication is error-prone, and the infection is characterized by its quasi-species nature. Antiretroviral treatment with such compounds as zidovudine (AZT), zalcitabine (ddC), didanosine (ddI), stavudine (d4T), and lamivudine (3TC) select for quasispecies variants that are resistant to these compounds (1). The detection of these variants is clinically important because they may affect the outcome of the treatment (2).

[Update on hepatitis C virus: its variability and the implications]. / Mise au point sur le virus de l'hépatite C: variabilité et implications.

Hepatitis C virus (HCV) is the main etiologic factor of post-transfusional and sporadic hepatitis, called non-A non-B in the past. These infections are characterized by a very high number of chronic carriers always with a persistent viral increase, but often at a slow pace. The seriousness of liver disease differs from one individual to another, varying from an asymptomatic form with minor or no liver injuries, to cirrhosis and hepatocellular carcinoma. Physiopathological mechanisms involved in liver injuries are still poorly understood. The direct role of immune response and of possible genetic factors is still under study. This review aims at summing up the discovery of HCV, its structure, and its variability in the various genome regions in the same individual and from one individual to another. The different methods and techniques to analyze this variability are also reviewed, as well as the various suggested ways of classifying the different types. The geographical distribution and both clinical and biological consequences of this variability are also discussed.

Evaluation of an affordable HIV-1 virological failure assay and antiretroviral drug resistance genotyping protocol.

HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDR(ultralight)). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS EasyQ v1.2, Roche) with an R(2) of 0.99. Accuracy studies illustrated standard deviations of <1 log RNA copies/ml for plasma and DBS ARTA-VFA results compared to the reference method. The ARTA-VFA's intended use was to deliver qualitative results either < or >5000 RNA copies/ml. No significant differences in the proportion of results < or > either the 5000 RNA copies/ml or 1000 RNA copies/ml cut-off were noted for plasma indicating either cut-off to be useful. Significant differences were noted in these proportions when DBS were used (P=0.0002), where a 5000 RNA copies/ml cut-off was deemed more appropriate. The sensitivity and specificity of the ARTA-VFA with plasma were 95% and 93% and 91% and 95% for DBS using a 5000 RNA copies/ml cut-off. The ARTA HIVDR(ultralight) assay was reliable for plasma and DBS samples with a viral load >5000 RNA copies/ml, with amplification and sequencing success rates of 91% and 92% respectively for plasma, and 95% and 80% respectively for DBS. HIVDR profiles for plasma and DBS were 100% concordant with the reference assay. This study evaluated a previously described combination of two assays potentially useful in assessing HIV-1 virological failure and resistance, showing good concordance with reference assays. These assays are simple to perform and are affordable, viable options to detect virological failures in certain resource limited settings. The assays' compatibility with DBS sampling extends the access of HIV-1 virological monitoring to more remote settings.

Cloning and phylogenetic analysis of the core, E2, and NS3/NS4 regions of the hepatitis C virus type 5a.

By means of the Line Probe Assay, a hepatitis C virus type 5a infected serum from a Belgian patient was selected. The complete core region (573 bp), the carboxyterminal part of E1 and the aminoterminal part of E2/NS1 (661 bp), and an epitope containing region in NS3-NS4 (1452 bp) were cloned and sequenced. The deduced amino acid sequence revealed type-specific variations in regions of core and NS4 which were previously recognized as evoking a type-specific antibody response. In addition, the aminoterminal region of E2 showed high variability when compared with sequences of type 1, 2, and 3. Phylogenetic analysis showed separate branching of this isolate. The analysis in the core region was not conclusive for some of the strains included and should therefore be carried out in combination with the NS5B region.

Persistence of viral replication after anti-HBe seroconversion during antiviral therapy for chronic hepatitis B.

BACKGROUND/AIMS: Hepatitis B virus genome mutants may be selected during the immune-mediated clearance of infection or during long-term nucleoside analog administration and may escape both antiviral pressures. The pattern of anti-HBe seroconversion was analyzed in patients receiving new nucleoside analogs, lamivudine or famciclovir, in comparison with patients treated with interferon alpha. METHODS: Eighteen consecutive patients who seroconverted to anti-HBe were included in the study. Serial serum samples were studied with the quantitative determination of HBV DNA by the branched DNA assay (Chiron) and by a quantitative PCR assay (Roche diagnostics), determination of pre-S1 Ag, the genetic analysis of the viral genome with the determination of pre-core promoter or pre-core region mutations with a line probe assay (Innogenetics) and, in selected samples of polymerase gene mutations. RESULTS: The quantitative PCR assay was found to be more sensitive than the bDNA assay, allowing a 25-log decrease in viral DNA levels to be demonstrated after anti-HBe seroconversion. Viral persistence after anti-HBe seroconversion induced by interferon, lamivudine or famciclovir, was often associated with circulating HBV genomes harboring mutations in the precore promoter. The clinical significance of these findings was demonstrated by the observation of reversion to HBeAg in two patients treated with interferon and one with lamivudine. CONCLUSION: Persistence of significant levels of viremia that are not detected by the branched DNA assay may be observed after anti-HBe seroconversion. A precise monitoring of viremia levels with more sensitive assays and HBV mutant strains is warranted in patients undergoing antiviral therapy.

Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and nonstructural 5B regions and identification of five additional subtypes.

Genotyping of hepatitis C virus-positive sera by means of a line probe assay indicated that < 3% of European samples, but up to 30% of Gabonese sera, could not be classified as either 1a, 1b, 2a, 2b, 3a, 3b, 4c, 5a, or 6a. Such samples were analyzed in the 5' untranslated region and in the nonstructural 5 (NS5) region. Classification based on phylogenetic analysis of the commonly used 222-bp-long NS5B region was possible for most but not all of the selected sera. Therefore, the core/envelope 1 region (579 bp) and a larger NS5B (340 bp) region were also analyzed. Only the phylogenetic analysis of the 340-bp NS5B region of these newly identified and published isolates provided unambiguous classification into types and subtypes. Furthermore, unequivocal evidence for four subtypes in type 2 and eight subtypes in type 4 was provided. A specific recognition sequence in the 5' untranslated region was observed for every newly identified subtype. Based on 1830 pair-wise comparisons in NS5B, isolates belonging to the same subtype showed evolutionary distances of < 0.127 and isolates of the same type exhibited evolutionary distances of < 0.328. These phylogenetic border distances can be conveniently used for classification of hepatitis C virus isolates into types and subtypes.

Analysis of the putative E1 envelope and NS4a epitope regions of HCV type 3.

Using a Line Probe Assay, type 3 HCV genotype-infected sera were selected from Brazilian blood donors. The partial nucleotide sequences of the core/E1 and NS4a epitope-containing regions and the NS5b typing region were determined. The E1 region had a nucleic acid homology of only 61 to 65% with the type 1 prototype genomes, and 56 to 58% homology with the type 2 prototype HCV genomes. Similar homologies were also found for the NS4a epitope region and for NS5. Furthermore, the deduced amino acid sequence of type 3 NS4a was used to generate synthetic peptides which were strongly reactive with human HCV-infected sera which were previously determined as anti-NS4 negative, indicating that a type-specific antibody response to the NS4a protein may exist.

Second-generation line probe assay for hepatitis C virus genotyping.

Because of the enormous variability of hepatitis C virus (HCV), the development of reliable genotyping assays is a formidable challenge. The optimal genotyping region appears to be the 5' untranslated region (UR) because of high conservation within, but considerable variability between, genotypes. In this study, 21 probes dispersed over seven variable 5' UR areas were applied to a line probe assay (LiPA) and used to analyze 506 HCV-infected sera from different geographical regions representing a multitude of subtypes. At least 31 different reactivity patterns emerged, with 404 (80%) of 506 distributed over 11 prototype patterns, in general corresponding to subtypes 1a, 1b, 2a/2c, 2b, 3a, 5a, and 6a and several type 4 subtypes. Subtyping specificity ranged from 97% in Hong Kong to 90% in Europe but was only 11% in West Africa, while typing specificity was always 100% when samples from Vietnam were excluded. In a second evaluation, the subtype prediction by LiPA of 448 GenBank 5' UR HCV sequences was scored. Of the 58 theoretically predicted patterns, 321 sequences (72%) were covered by the 11 prototype patterns. We concluded that (i) the selected probes detected the corresponding signature motifs in the seven variable regions with 100% reliability; (ii) these motifs allowed correct type interpretation of samples collected worldwide, with the exclusion of Vietnam, Thailand, or Vietnamese patients residing in European hospitals; and (iii) subtyping specificities vary according to geographical region, with 11 prototype subtyping patterns identifying the majority of samples from Europe and the Americas. These results indicate that the LiPA is a reliable assay applicable to routine typing and subtyping of HCV specimens.

Comparative analysis of two assays for genotyping hepatitis C virus based on genotype-specific primers or probes.

BACKGROUND/AIMS: The importance of determining the hepatitis C virus genotype has increased since several authors have described a good correlation between hepatitis C virus genotype and response to interferon treatment or to disease severity. It is thus of particular importance to develop reliable assays for hepatitis C virus genotyping. METHODS: We have comparatively analyzed hepatitis C virus genotypes of 208 French and Italian chronically infected patients using genotype-specific primers polymerase chain reaction in the capsid region and a genotype-specific probe-based assay in the 5' untranslated region (LiPA). RESULTS: We found a good concordance between the two assays for the prevalent genotypes in our regions (145/174). The nucleotide sequences in the 5'UTR and capsid domain were investigated to determine the molecular basis of some discordant results. This analysis showed that the genotype-specific probe-based assay gave more consistent results, probably because this technique is based on several probes distributed along the 5'UTR to reveal each genotype. In contrast, the low variability of 5'UTR did not always permit classification of the hepatitis C virus genotype 1 subtypes. We also found some problems, using genotype-specific primers polymerase chain reaction, for less represented genotypes; in particular, in the presence of type 2a/III we obtained more discordant results. This observation is of importance in view of the potential association of this genotype with mild liver disease and a good response to interferon-a treatment.

Line probe assay for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy.

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.

A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness.

The hepatitis B virus (HBV) genotype was determined in a total of 121 plasma samples collected in France and the US from patients chronically infected with HBV. HBV genotype A was predominant in this collection, appearing in 66 samples (54%), while genotypes B, C, D, E and F occurred in 4 (3%), 14 (12%), 23 (19%), 1 (1%) and 0 (0%) of samples, respectively. However, the genotype of a total of 13 (11%) samples (2 from France, 11 from the US) could not be determined with the methodology used. Sequence analysis, and subsequent phylogenetic analysis of the complete genome and the individual open reading frames, showed that the virus isolate from these samples was 3248 bp long and, phylogenetically, did not cluster with any of the known genotypes. This strain was provisionally called HBV genotype G. Virus isolates that were obtained from geographically separated regions like France and the US were closely related to each other. All virus strains analysed contained some characteristic differences when compared to genotype A: a translational stop codon at aa 2 and 28 of the preCore region; a 36 nt (12 aa) insert in the amino-terminal part of the Core antigen (HBcAg); a 2 aa deletion in the carboxy-terminal part of HBcAg; and a 1 aa deletion in the preS1 open reading frame. The deduced amino acid sequence of HBsAg suggests that this newly discovered genotype G strain belongs to serological group adw2.