of genes involved in latency and reactivation of Pseudorabies virus,
use in biological containment study of viral genomes in pigs
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.
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.
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.
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.
Boutin P., Arnauld C., Thiery R., Costa J.M., Vidaud M., Jestin
A., Chemiluminescent detection of amplified pseudorabies
virus gp50 DNA with immobilized probes on microtiters wells.
Acta Veterinaria Hungarica, 42,
1994, pp. 385-387.
Thiery R., Boutin P., Arnauld C., Jestin A., Pseudorabies
virus latency: a quantitative approach by polymerase chain reaction.
Acta veterinaria Hungarica, 42,
1994, pp. 277-287.
Foulon T., Kaido T., Fuchs W., Rziha H.J., Sheldrick P., A
bovine cell line stably expressing porcine tumor necrosis factor
alpha (TNF-alpha): growth properties and permissivity for pseudorabies
Acta Veterinaria Hungarica, 42, 1994, pp. 195-203.
October 1991 March 1994
Federal Research Centre for Virus Diseases of Animals
Institut de Recherche sur le Cancer
I.M. Correia de Almeida
National Veterinary Research Laboratory