Detection of vesicular stomatitis virus RNA and its defective-interfering particles in individual mouse brains

To develop a highly sensitive and direct assay for defective interfering (DI) particles of vesicular stomatitis virus (VSV), we reverse transcribed RNA from DI particles and cloned the DNA in pBR322 and used it as hybridization probes. At the lower limit, cDNA of about 850 nucleotides detected 150 pg of VSV RNA. For differentiation of hybridizable sequences found in the RNA of DI particles from complementary or identical sequences in the L mRNA or standard genomic RNA of VSV, RNA obtained from mouse brains was first separated by size, blotted onto nitrocellulose, and then hybridized to in vitro-labeled cDNA probe. Genomic VSV, DI, or L mRNA sequences from one-half of the brain of an infected mouse were detectable, whereas uninfected mice failed to react with this specific probe. When mice were infected intranasally with 10(8) PFU of standard VSV, most of them died between days 6 and 7, and the detection of standard genomic RNA correlated with paralysis and death. DI RNA was not detected in these mice. When mice were infected with 10(8) PFU of standard VSV together with an equivalent amount of DI particles, similar results were obtained. When fewer DI particles were inoculated together with standard virus, significant protection of mice occurred together with the detection of DI RNA. These results indicate that DI particles are protective in vivo and that the details of the virus-host interaction may resemble the cyclic growth patterns in cell cultures for standard VSV and its DI particles.