ABSTRACT
The objective of this study was to compare the performance of the Idylla™ Respiratory (IFV-RSV) panel to the GeneXpert Xpert® Flu/RSV assay and establish the performance of a midturbinate swab compared to nasopharyngeal sampling. Considering GeneXpert® assay as imperfect reference standard, a positive percentage agreement between both assays of 98-100% for influenza A and 96-99% for influenza B could be calculated when 354 nasopharyngeal and 325 midturbinate swabs were retrospectively analyzed. Comparing midturbinate samples to nasopharyngeal specimens of 321 subjects, positive percentage agreement varied from 42% to 94% depending on both target virus and assay used. Negative percentage agreements ranged from 98% to 100% for both methods and sample type comparison. The Idylla™ assay showed excellent performance compared to the GeneXpert® assay for the detection of influenza virus. The study also showed a slightly better performance for nasopharyngeal sampling compared to the use of a midturbinate swab.
Subject(s)
Influenza, Human/diagnosis , Molecular Diagnostic Techniques/methods , Nasal Cavity/virology , Nasopharynx/virology , Orthomyxoviridae/isolation & purification , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus, Human/isolation & purification , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Retrospective Studies , Specimen Handling/methods , Young AdultABSTRACT
A hepatitis C virus (HCV) replicon-based protease phenotyping assay has been developed that allows determining the susceptibility of a patient's HCV protease sequence to HCV protease inhibitors. In brief, HCV protease sequences amplified from clinical samples are cloned in a transient HCV genotype 1b replicon backbone, containing a luciferase reporter gene. These protease chimeric replicons are replication-competent when electroporated into susceptible Huh7-Lunet cells. Replication can be quantified by measuring the enzymatic activity of the luciferase protein. This assay is reproducible and robust, and has a high overall success rate for determining the phenotypic susceptibility of HCV genotype 1a and 1b patient-derived protease domains to HCV protease inhibitors. In addition, the HCV genotype 1b protease shuttle backbone also supports efficient replication of HCV genotype 4 protease sequences.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepacivirus/physiology , Microbial Sensitivity Tests/methods , Protease Inhibitors/pharmacology , Virus Replication/drug effects , Cell Line , Cell-Free System , Cloning, Molecular , Genetic Vectors/genetics , Hepacivirus/isolation & purification , Humans , Polymerase Chain Reaction , Transcription, Genetic , Viral Nonstructural Proteins/antagonists & inhibitorsABSTRACT
Genotypic testing based on subtype-specific amplification and population Sanger sequencing for two nonstructural (NS) protein-coding regions, the NS3/4A protease and the NS5B polymerase, of the hepatitis C virus (HCV) genome is described here. The protocols include the molecular steps for RNA extraction, one-step RT-PCR followed by inner PCR and population Sanger sequencing, to obtain the sequence information of the target regions from the clinical isolates of HCV subtypes 1a and 1b, which can be used to detect any sequence change in the viral genome as for example caused by the development of drug resistance in these two common viral targets.
Subject(s)
Drug Resistance, Viral/genetics , Genotype , Hepacivirus/drug effects , Hepacivirus/genetics , Microbial Sensitivity Tests/methods , Viral Nonstructural Proteins/genetics , Cell Line , Humans , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
BACKGROUND: Hepatitis C virus (HCV) genotyping and accurate subtype determination is becoming increasingly important to better understand viral evolution, the development of resistance to STAT-C, and possibly even for the treatment and management of chronic HCV-infected patients. METHODS: A subtyping assay based on a 329-bp sequence of the NS5B region, together with an automated subtype interpretation tool was developed. Clinical samples of the six major genotypes were used to assess assay performance characteristics. RESULTS: The NS5B BLAST-based subtyping assay showed clinical sensitivity for amplification of 89% (n=603 random samples). Assessment of analytical sensitivity of amplification for genotypes 1, 2, 3 and 4 revealed a suitable performance for high viral load samples and decreased only with low viral loads. The results were 100% and 99% accurate at the genotype and subtype level, respectively. A concordance of 97% on genotype level and 62% on subtype level was observed by comparison with subtype results from 5' non-coding-based assays with a panel of 276 isolates. CONCLUSIONS: The HCV NS5B subtyping assay has been validated for research use. Based on its performance, it is the method of choice in cases where subtype rather than genotype information is needed.
Subject(s)
Hepacivirus/classification , Protein Subunits/genetics , Viral Nonstructural Proteins/genetics , Automation , Genotype , Hepacivirus/genetics , Hepacivirus/isolation & purification , Humans , Phylogeny , RNA, Viral/genetics , Reagent Kits, Diagnostic , Sequence Analysis, RNA , Viral LoadABSTRACT
Chronic hepatitis B virus (HBV) infection is a major cause of liver disease. Only interferon-alpha and the nucleosidic inhibitors of the viral polymerase, 3TC and adefovir, are approved for therapy. However, these therapies are limited by the side effects of interferon and the substantial resistance of the virus to nucleosidic inhibitors. Potent new antiviral compounds suitable for monotherapy or combination therapy are highly desired. We describe non-nucleosidic inhibitors of HBV nucleocapsid maturation that possess in vitro and in vivo antiviral activity. These inhibitors have potential for future therapeutic regimens to combat chronic HBV infection.
Subject(s)
Acetylcysteine/analogs & derivatives , Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Nucleocapsid/metabolism , Pyridines/pharmacology , Pyrimidines/pharmacology , Triazoles/pharmacology , Acetylcysteine/pharmacology , Amino Acid Substitution , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Binding Sites , Capsid/metabolism , DNA Replication/drug effects , DNA, Viral/biosynthesis , Half-Life , Hepatitis B Virus, Duck/drug effects , Hepatitis B Virus, Duck/metabolism , Hepatitis B virus/physiology , Humans , Mutation , Pyridines/chemistry , Pyridines/metabolism , Pyrimidines/chemistry , Pyrimidines/metabolism , Recombinant Proteins/metabolism , Stereoisomerism , Triazoles/chemistry , Triazoles/metabolism , Tumor Cells, Cultured , Viral Core Proteins/chemistry , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Virus Assembly/drug effects , Virus Replication/drug effectsABSTRACT
In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a C-terminally truncated HBc (HBc) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBc mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain [J Gen Virol 1997;78:2049-2053]. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein. To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of 'non-mosaic' HBc fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein. Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBc particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa. The mosaic particles formed exhibited PUUV-N protein antigenicity. Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1-114, aa 1-213 and aa 340-433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1-114 or aa 1-213 of the N protein. Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated. In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and 'problematic', e.g. hydrophobic, hantavirus sequences.
