Date Full Report Received


Date Abstract Report Received



Primary Investigator:

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major virus threats facing the swine industry in the USA and worldwide. Whilst the introduction of vaccines has been moderately successful in the control of the disease, new strains are constantly emerging, especially from Asia.  Live virus vaccines have been shown to be the only type of vaccine capable of establishing protective immunity against PRRSV.  Design of these vaccines has been greatly helped by the establishment of the ability to genetically manipulate the virus in the laboratory setting – with the goal of altering the growth properties and/or ability of the virus to cause disease.  The purpose of this project was to investigate the functioning of a virus protein, called the nucleocapsid (N) protein, which is critical for the virus life cycle.  The rationale of the project was to define the biological properties of this protein in order to attenuate virus growth – and hence present candidate vaccines.  Particular focus was placed on interfering with the ability of the N protein to interact with the virus genetic material (composed of a molecule called RNA).  Towards this end, the research was split into three main Objectives and a contingency Objective:

Objective One was to construct a suite of recombinant PRRSV with viral RNA binding mutations in the N protein and to refine our knowledge of RNA binding.  This objective involved refining RNA binding studies from Year One of the project and constructing a range of recombinant viruses whose N proteins were able to bind RNA with different efficiencies to that of N protein from wild-type virus – based on data from Year 1.  The prediction being that these viruses should show a gradation of growth attenuation.

Objective Two was to characterize recombinant viruses in cell culture and compare their biology to wild type and a vaccine virus strain.  The goal being to assess the degree of attenuation in these viruses compared to virus that causes disease and a similar vaccine strain.

Objective Three was to assess the stability of recombinant viruses. One of the problems associated with live-attenuated vaccines of RNA viruses (including PRRSV) is that they revert back to virulence, and this objective was to assess the stability of recombinant viruses that could be grown and recovered.

Objective Four was a contingency objective to improve the genome stability of recombinant viruses, in order to prevent reversion to wild type.

As detailed in the objectives several recombinant viruses were successfully constructed whose N proteins showed alterations in biological activity – in terms of binding to viral RNA, these focused on mutant N proteins that when expressed in isolation had less binding when compared to the normal wild type N protein.  The rationale at this stage was to introduce enough changes in the N protein to prevent reversion at a latter stage.  Comparison of virus biology between the recombinant viruses and the wild type virus and the vaccine virus indicated that the virus with the ability to bind viral RNA with less efficient that wild type grew less well.  Those recombinant viruses which had the capability to bind RNA with approximately 15% and 25% did not effectively grow in cell culture, indicating that these changes were lethal for the virus.  Extensive growing of the virus with approximately 80% RNA binding activity indicated that this property remained conserved, and no reversion to wild type occurred, thus the recombinant mutant virus was stable in cell culture.  The major implications for industry are two fold.  First, the biological properties of N protein can be genetically manipulated in order to attenuate virus growth – thus alterations of N protein function can be used in the design of live recombinant growth attenuated vaccines.  Second, because this project has thoroughly defined the role of N protein binding to the viral genome RNA and the three-dimensional structure of the N protein is known, the possibility now arises that chemotherapeutic intervention against PRRSV can be considered and developed for emergency applications, such as the sudden emergence of virulent strains not immediately accessible to containmen through a vaccine.