A comparison of different genomic probes in the detection of virus-specified RNA in Orbivirus-infected cells

Abstract

Different 32P-labelled genomic probes of bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and equine encephalosis virus (EEV) were compared with respect to the detection of virus-specified RNA in infected cells. The probe derived from the genome segment that encodes nonstructural protein NS1 was found to be the most sensitive, detecting virus-specified RNA in glutaraldehyde-fixed cells as early as 2-3 h p.i. This comparison was based on the observation that the NS1 gene probe required a smaller number of infected cells to produce a positive hybridization signal than the other nucleic acid probes. The only exception was the EHDV NS2 gene probe which appeared to be as sensitive as the NS1 gene probe. The advantage of using the NS1 gene probe was particularly evident in the analysis of cells infected at very low multiplicities of infection. At a multiplicity of infection of 1 x 10(-5) plaque forming units/cell, virus-specified RNA could be detected 48 h after infection. The greater sensitivity of the NS1 gene-specific probe is ascribed to the fact that its target, the NS1 mRNA, is transcribed more frequently than the other target viral mRNAs. The major application of the cell-hybridization method is the rapid detection of small quantities of infectious virus particles.