Evaluation of a prototype HCV NS5b assay for typing strains of hepatitis C virus isolated from Tunisian haemodialysis patients.

Hepatitis C virus (HCV) strains isolated from 68 haemodialysis Tunisian patients exhibiting chronic infection were genotyped targeting the NS5b region of the HCV genome using a prototype assay developed by Bayer HealthCare-Diagnostics (TRUGENE NS5b HCV). The overall results were compared to those obtained with another assay of the same company based on sequencing of the 5' non-coding region (TRUGENE HCV 5'NC genotyping kit). All strains could be typed by the 5'NC typing kit, but only 62 (91; 2%) by the NS5b prototype assay. All the 62 strains typed by both methods exhibited the same pattern at the type level: 57 were type 1, 3 were type 2, and 2 were type 4. At the subtype level, eight strains that gave undetermined results by the 5'NC kit were successfully typed by the NS5b kit; eight additional strains exhibited discrepant results. The overall agreement between the two assays was 74.2% at the subtype level. In conclusion, the NS5b region appears to be much more accurate than the 5'NC region to subtype HCV strains, especially in those isolated from patients attending haemodialysis centres where the subtype distribution suggests frequent nosocomial transmissions.

Prevalence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-associated mutations and polymorphisms in NNRTI-naïve HIV-infected patients.

BACKGROUND: The efficacy of treatment containing nonnucleoside reverse transcriptase inhibitors (NNRTIs) could be compromised in NNRTI-naïve patients already harboring a virus resistant to NNRTIs. On the contrary, hypersusceptibility to NNRTIs in patients having failed nucleoside reverse transcriptase inhibitor (NRTI)-containing regimens has been described and has been associated with improved outcome. METHOD: We assessed the prevalence of NNRTI resistance-associated mutations or polymorphisms in 146 antiretroviral-naïve patients and in 181 HIV-infected patients who were given an NNRTI-based regimen. We phenotypically evaluated the NNRTI susceptibility of 41 strains presenting with amino acid substitutions at positions involved in NNRTI resistance. RESULTS: In the 268 genotypically analyzable samples, the overall prevalence of NNRTI resistance-associated mutations was 2% (6/268 patients). The prevalence of strains with amino acid substitutions at reverse transcriptase (RT) gene positions (A98, K101, K103, V106, V108, V179) involved in NNRTI resistance was 15%. Hypersusceptibility to NNRTI was rare (2%, 1/41) in those samples. RT substitutions at positions involved in NNRTI resistance were not associated with a significantly worse virologic outcome in NNRTI-treated patients. Our understanding of small shifts in IC50 values (higher or lower) toward NNRTI is very limited. The significance of many RT mutations on NNRTI susceptibility is not clear. CONCLUSION: In contrast to resistance mutations, RT substitutions at positions involved in NNRTI resistance are frequent. They are not associated with a worse virologic outcome or with decreased phenotypic susceptibility to NNRTIs. It may be prudent not to rule out the use of NNRTIs in patients with small shifts in IC50 values or poorly understood mutations.

Genotyping of hepatitis C virus isolates using CLIP sequencing.

Determination of hepatitis C virus (HCV) genotypes and subtypes has become increasingly important for the clinical management and prognosis of HCV infections. The aim of the present study was to assess the specificity and reliability of a newly developed, commercially available HCV genotyping kit (TRUGENE HCV 5'NC genotyping kit). This technique utilizes PCR fragments previously generated by the diagnostic Roche AMPLICOR HCV test, which are subsequently subjected to simultaneous PCR amplification and direct sequencing (CLIP sequencing) of the 5' noncoding region (5'NCR). HCV isolates from 100 randomly chosen patients were genotyped by both the TRUGENE HCV 5'NC genotyping kit and DNA enzyme immunoassay (DEIA). Typing results obtained by both methods were in complete concordance in 91% of the cases. HCV RNA from the samples with discordant genotype assignment in both assays was additionally amplified with primers from the HCV core and NS5B regions. Phylogenetic analysis of the obtained sequences supported the results obtained from DEIA in six cases and CLIP sequencing in two cases. In the former six cases, the TRUGENE HCV 5'NC genotyping kit could not correctly differentiate between subtypes of genotypes 1 and 2 due to the high conservation of the 5'NCR. However, since there was not any misclassification between HCV genotypes 1 and non-1 types, the results obtained with this system are, in general, reliable and can be used in clinical practice. The TRUGENE HCV 5'NC genotyping kit in our hands proved to be a fast and convenient technique that might be an attractive option for HCV genotyping in laboratories already using the Roche AMPLICOR HCV test for diagnostic reverse transcription-PCR.

Stabilization of methionine-rich protein in Saccharomyces cerevisiae: targeting of BZN protein into the peroxisome.

We have constructed a gene coding for the 12-kDa intermediate form of the 2s methionine-rich protein from Bertholletia excelsa seeds. This protein, expressed intracellularly in yeast, is characterised by a 20-min half-life. By adding 11 amino acids corresponding to the peroxisome-targeting sequence (PTSc) of luciferase, we have significantly increased its half-life. This stabilization allowed accumulation of the BZN protein into the peroxisome as judged by cell fractionation. Accumulation of the 12-kDa protein results in a significant increase of the total methionine content in yeast cells (30%) indicating that such a microorganism could represent a practicable protected shuttle for an animal-feed additive.

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat.

Mini-Mu transduction: cis-inhibition of the insertion of Mud transposons.

Mud (mini-Mu) transposons are defective phage Mu genomes that conserve the Mu ends. The transduction of Mud transposons is strictly dependent on Mu complementation, inefficient, and affected by modifications in the Mud internal sequences. The transduction of Mud transposons depends on transposition, which appears to be low, relative to wild-type Mu. Insertions of Mud into a plasmid can be frequently recovered among transductants; new Mud insertions into plasmids that already have both Mu ends, or just one, are rarely found. This suggests that the presence of Mu ends "immunizes" the plasmid against further insertion. This phenomenon may be similar to the transposition immunity of Tn3.

lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid.

Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL-region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression.