Discrimination within epitope specific antibody populations against Classical swine fever virus is a new means of differentiating infection from vaccination.

Serological differentiation between infection and vaccination depends on the detection of pathogen specific antibodies for an epitope that is modified or lacking in a vaccine. Here we describe a new assay principle that is based on differences in the binding properties of epitope specific antibodies. C-DIVA is a potent Classical swine fever vaccine candidate that differs from the parental C-strain life attenuated vaccine in the highly immunogenic TAVSPTTLR epitope by the deletion of two and the mutation of one amino acid (TAGSΔΔTLR).

Quantitative detection of hepatitis B virus DNA by real-time nucleic acid sequence-based amplification with molecular beacon detection.

We have developed a hepatitis B virus (HBV) DNA detection and quantification system based on amplification with nucleic acid sequence-based amplification (NASBA) technology and real-time detection with molecular beacon technology. NASBA is normally applied to amplify single-stranded target RNA, producing RNA amplicons. In this work we show that with modifications like primer design, sample extraction method, and template denaturation, the NASBA technique can be made suitable for DNA target amplification resulting in RNA amplicons.

Characterization of the quantitative HCV NASBA assay.

A quantitative nucleic acid sequence-based amplification assay (NASBA-QT) for detection of hepatitis C virus RNA (HCV-RNA) was evaluated and compared with the HCV branched-DNA (bDNA) assay (Chiron Corporation) and the HCV MONITOR assay (Roche Diagnostic Systems). For this evaluation five panels were designed: (1) serial dilutions of genotype 1b in-vitro HCV-RNA; (2) standards of in-vitro HCV-RNA genotypes 1a, 1b, 2, 3, 4, and 5; (3) a proficiency panel consisting of 12 HCV-RNA positive plasma samples of different genotypes and HCV-RNA concentrations and a genotype 1a and 1b 3-fold dilution series; (4) a panel of 67 HCV-RNA positive plasma samples obtained from patients with HCV infection and (5) an HCV-RNA positive control sample, diluted 50-fold in 25 different HCV-RNA negative plasma samples. The quantitative detection limit was found to be 10(3) copies per 100 microl and the qualitative detection limit 10(2.

Stability of hepatitis C virus RNA during specimen handling and storage prior to NASBA amplification.

The influence of different anticoagulants and pre-amplification storage conditions on the stability of hepatitis C virus (HCV)-RNA, as detected by the quantitative HCV NASBA assay (NASBA-QT), was studied. The HCV-RNA load remained stable for at least 15 months when serum or plasma samples (EDTA and heparin) were directly frozen at -70 degrees C in lysis buffer. At 4 degrees C, the HCV-RNA load in serum or plasma stored with lysis buffer did not decline for at least 14 days.

Serological and molecular analysis of hepatitis C virus envelope regions 1 and 2 during acute and chronic infections in chimpanzees.

Acute and chronic Hepatitis C virus infections were investigated retrospectively in chimpanzees that had been infected from a single source. Anti-E1 and anti-E2 were detected in two of three chimpanzees with a chronic infection, but were first detected 1 to 2 years after inoculation. Sequence evolution of the E1 region in three animals over a period of 9 to 11 years revealed a mutation rate of 1.