Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus

Biogenesis of the Semliki Forest virus RNA replication complex.

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typic...

NELE TAMBERG Studies on Semliki Forest virus replication and pathogenesis

Guidelines for quantitative rt-PCR.

Something I notice often in supervising new graduate students is their frustration with the irreproducibility of their biological data. For example, I am asked frequently ‘‘how many biological replicates do I need for qRT-PCR’’? The answer, of course, lies first with the students themselves. They need as many replicates as will persuade them of the validity of their observations. However, for m...

Detection and quantitation of bovine respiratory syncytial virus using real-time quantitative RT-PCR and quantitative competitive RT-PCR assays.

A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad's iCycler iQ. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovi...

Properties of non-structural protein 1 of Semliki Forest virus and its interference with virus replication

Semliki Forest virus (SFV) non-structural protein 1 (nsP1) is a major component of the virus replicase complex. It has previously been studied in cells infected with virus or using transient or stable expression systems. To extend these studies, tetracycline-inducible stable cell lines expressing SFV nsP1 or its palmitoylation-negative mutant (nsP16D) were constructed. The levels of protein exp...