Background and objectives

The alpha-herpesvirus Pseudorabies virus (PRV, Suid, herpes 1) has the propensity to establish a latent infection in the natural host, pig. This latent infection can be reactivated and affects both neural tissues and lymphocytes. Available vaccines including gene technology derived live vaccines do not prevent latency, and live vaccines can also establish a latent infection.

This research project aimed to identify the viral genes influencing the establishment of latent infections and reactivation from latency of Pseudorabies virus in its natural host, pigs. The major objective was a detailed analysis of the biosafety of PRV mutants with modifications specifically affecting the latency-associated functions. Latency associated transcripts (LATs), that are RNA molecules, were identified, characterised and the molecular organisation of the corresponding genomic region was elucidated. A thorough understanding of the latency phenomenon may allow the construction of novel and safer live vaccines.

Pigs.

Approach and methodology

After DNA sequencing and transcriptional analysis, various relevant parts of the LAT were inserted into plasmids. Fusions to the functional promoterless luciferase gene used as an indicator for cis-acting promoter elements were constructed. Diverse cell lines were transformed with these plasmid constructs and transient luciferase expression was assayed. Putative latency associated promoters (LAP) were thereby identified.

Genetic variants of PRV were selected to examine the kinetic parameters of PRV neuropathogenesis.

A highly sensitive, specific and quantitative PCR method was developed to detect small amounts of PRV virus in infected tissues.

Main findings and outcome

An optimal transfection protocol was established for measuring transient luciferase expression in the different cell lines. We found comparable promoter activity in four different epithelial cell lines (of porcine, bovine and simian origin) and mouse neuroblastoma cell lines. A 270 base pair fragment was obtained which showed maximum in vitro promoter activity, and is thus a LAP element. Starting with the lacZ-labelled wild-type PRV we constructed four different LAT-deletion mutants. A specific RT-PCR was established and used to analyse these mutants. All LAT-/LAP- mutants express a LAT RNA which is spliced in exactly the same way as the wild-type virus. These findings indicate the existence of an additional promoter. Various virological studies were conducted. Viral DNA and mRNA was identified by in situ hybridisation techniques with cDNA probes labelled by multipriming, or cRNA probes synthesised by reverse transcription.

Immunohistochemistry was used to study viral antigens, and the production of infectious progeny virus was also investigated. Microglia, Schwann cells and DRG fibroblastoid cells were all productively infected. A highly specific quantitative PCR method was developed based on the use of an internal standard which is added at known concentrations to the components of the PCR and co-amplified with the target DNA. Pigs were infected with a PRV deletion mutant (from which the putative LAP had been totally deleted) or with wild-type virus, and studied at various times after infection. Viral DNA of both strains was detected by quantitative PRV in several neural tissues. The amounts of virus present in latently infected lymphocytes and in trigeminal ganglia do not appear to differ significantly between animals infected with the wild-type and those infected with the deletion mutant. To produce biologically active porcine TNF-alpha, its gene was inserted into a shuttle vector downstream from the RSV LTR promoter. The construct was introduced into MDBK cells by transfection. A stable transformed cell was obtained and secreted 20 to 30 pg/ml of active TNF-alpha into the growth medium after five days in culture.

Conclusions

A limited region of the PRV genome was identified which appears to be transcribed mostly during the latent stage of infection. Promoter activity was demonstrated, and the promoter element was isolated. The first defined LAT-mutants of PRV, that is viruses with modifications of the latency associated genes, have been obtained. An analysis of a PRV mutant totally lacking in the LAT promoter, indicated that the presence of the LAT promoter is not necessary for the establishment of latency and does not influence the amounts of virus found in infected animals. From the methodological standpoint, one major achievement was the development of a quantitative PCR method allowing more accurate analysis of the pathogenic virus.