Effects of deletion of the early protein 0 gene of pseudorabies virus on the overall viral gene expression.

Real-time RT-PCR analysis was applied to evaluate the impact of deletion of the early protein 0 (EP0) gene of pseudorabies virus (PRV) on the global expression of the viral transcripts during lytic infection in cultured porcine kidney cells. Our analysis showed that EP0 exerted an inhibitory effect on the transcription of the PRV genes in the early stage of infection, and alternating stimulatory and inhibitory effects on the viral gene expressions in the late stage of infection. The data also suggested that a general function of EP0 might be to reverse the kinetics of expression of early viral genes.

Deletion of the virion host shut: off gene of pseudorabies virus results in selective upregulation of the expression of early viral genes in the late stage of infection.

A real-time RT-PCR technique was applied to evaluate the impact of deletion of the virion host shut-off (VHS) gene on the kinetics of pseudorabies virus gene expression. Selective suppression of early gene transcripts by the viral ribonuclease occurs after 4h of infection; while VHS protein appears to act non-selectively on the transcripts belonging in different kinetic classes in the first 2h of infection. VHS protein disrupts the close correlation between the transcription kinetics of the immediate-early 180 protein and the other pseudorabies virus transcripts.

The effects of viral load on pseudorabies virus gene expression.

Background: Herpesvirus genes are classified into distinct kinetic groups on the basis of their expression dynamics during lytic growth of the virus in cultured cells at a high, typically 10 plaque-forming units/cell multiplicity of infection (MOI). It has been shown that both the host response and the success of a pathogen are dependent on the quantity of particles infecting an organism. This work is a continuation of an earlier study 1, in which we characterized the overall expression of PRV genes following low-MOI infection.

Ex vivo infection of human embryonic spinal cord neurons prior to transplantation into adult mouse cord.

Background: Genetically modified pseudorabies virus (Prv) proved suitable for the delivery of foreign genes to rodent embryonic neurons ex vivo and maintaining foreign gene expression after transplantation into spinal cord in our earlier study. The question arose of whether human embryonic neurons, which are known to be more resistant to Prv, could also be infected with a mutant Prv. Specifically, we investigated whether a mutant Prv with deleted ribonucleotide reductase and early protein 0 genes has the potential to deliver marker genes (gfp and beta-gal) into human embryonic spinal cord neurons and whether the infected neurons maintain expression after transplantation into adult mouse cord.

Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay.

Background: Pseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection.

Results: In this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools.

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

Cerebral neurons involved in the innervation of both the adrenal gland and the ovary: a double viral tracing study.

Previous studies using the viral transneuronal tracing technique demonstrated central autonomic circuits involved in the innervation of the adrenal gland and the ovary. Since the pattern of infection of central nervous system structures is similar after virus inoculation of the adrenal gland and the ovary, and, on the other hand, it is well documented that the activity of the hypothalamo-pituitary-adrenal axis exerts an inhibitory effect on the reproductive system, we investigated whether there are neurons that are transneuronally connected both with the adrenal gland and the ovary. The central circuitry involved in the innervation of the left adrenal and the left ovary was studied in individual rats by dual transneuronal tracing using isogenic recombinant strains (BDG and DS-RED) of Bartha strain of pseudorabies virus.