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. We also observed that EP0 facilitated the development of correlations in the transcription kinetics between the immediate early 180 gene and the PRV transcripts, indicating that a major function of EP0 could be to modify the effects of the IE180 protein on the PRV transcriptome.

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 typical pattern of early gene expression was found to be altered in the VHS gene-deleted virus in that the production rates of their transcripts did not decline from 4h post-infection. This observation led us to put forward the hypothesis that the VHS protein may play a pivotal role in the switch from the early to the late stage of infection.

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. In the present study, we have addressed the question of whether viral gene expressions are dependent on the multiplicity of infection by comparing gene expressions under low and high-MOI conditions. RESULTS: In the present study, using a real-time RT-PCR assay, we address the question of whether the expression properties of the pseudorabies virus (PRV) genes are dependent on the number of virion particles infecting a single cell in a culture. Our analysis revealed a significant dependence of the gene expression on the MOI in most of these genes. Specifically, we found that most of the examined viral genes were expressed at a lower level at a low MOI (0.1) than at a high MOI (10) experiment in the early stage of infection; however, this trend reversed by six hour post-infection in more than half of the genes. Furthermore, in the high-MOI infection, several PRV genes substantially declined within the 4 to 6-h infection period, which was not the case in the low-MOI infection. In the low-MOI infection, the level of antisense transcript (AST), transcribed from the antiparallel DNA strand of the immediate-early 180 (ie180) gene, was comparable to that of ie180 mRNA, while in the high-MOI experiment (despite the 10 times higher copy number of the viral genome in the infected cells) the amount of AST dropped by more than two log values at the early phase of infection. Furthermore, our analysis suggests that adjacent PRV genes are under a common regulation. This is the first report on the effect of the multiplicity of infection on genome-wide gene expression of large DNA viruses, including herpesviruses. CONCLUSION: Our results show a strong dependence of the global expression of PRV genes on the MOI. Furthermore, our data indicate a strong interrelation between the expressions of ie180 mRNA and AST, which determines the expression properties of the herpesvirus genome and possibly the replication strategy (lytic or latent infection) of the virus in certain cell types.

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. RESULTS: The results revealed that the mutant Prv effectively infected human embryonic spinal cord neurons ex vivo and the grafted cells exhibited reporter gene expression for several weeks. Grafting of infected human embryonic cells into the spinal cord of immunodeficient (rnu-/rnu-) mice resulted in the infection of some of the host neurons. DISCUSSION: These results suggest that Prv is suitable for the delivery of foreign genes into transplantable human cells. This delivery method may offer a new approach to use genetically modified cells for grafting in animal models where spinal cord neuronal loss or axon degeneration occurs.

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. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners. CONCLUSION: Most (if not all) PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular R value. In principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems.

Herpesvirus-mediated delivery of a genetically encoded fluorescent Ca(2+) sensor to canine cardiomyocytes.

We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent Ca(2+) sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (I(to)), kinetics of the intracellular Ca(2+) transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the I(to) current was significantly changed and no major shifts occurred in parameters of [Ca(2+)](i) transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of 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. Dual-infected neurons were detected in the ventrolateral medulla, nucleus of the solitary tract, caudal raphe nuclei, A5 cell group, and hypothalamic paraventricular nucleus. The results indicate that there are neurons in the central nervous system that contribute to the transneuronal innervation of both the adrenal gland and the ovary. The data suggest a new type of interaction, i.e. interaction at cellular level that might be involved in regulatory processes integrating the functional activity of the two organs.