Biochemical and molecular analysis of structural proteins of porcine reproductive and respiratory syndrome virus

Abstract - Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the recently recognized Arteriviridae family within the genus Arterivirus, order Nidovirales, which also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). Mature viral particles are composed of an envelope, 50-72 nm in diameter, with a 20-30 nm isometric core enclosing a linear positive-stranded RNA genome of approximately 15 kb. The virions are assembled by the budding of preformed nucleocapsids into the lumen of the smooth endoplasmic reticulum and/or Golgi apparatus. The mature virions are then released by exocytosis. The viral genome contains eight open reading frames (ORFs) which are transcribed in the cells as a nested set of subgenomic mRNAs. The ORF1a and ORF1b situated at the 5' end of the genome represent nearly 75% of the viral genome and code for proteins with apparent replicase and polymerase activities. The major structural proteins consist of a 25 kDa envelope glycoprotein (GP ₅), an 18-19 kDa unglycosylated membrane protein (M), and a 15 kDa nucleocapsid (N) protein, encoded by ORFs 5, 6 and 7, respectively. In cells and virions, both M and GP ₅ occur in heterodimeric complexes linked by disulphide bonds. The expression products of ORFs 2 and 4 are also incorporated into virus particles as additional minor membrane-associated glycoproteins designated as GP ₂ and GP ₄, with M_r of 29 and 31 kDa, respectively. Apart from its nonstructural nature, recent reports indicate that the ORF3 product, a highly glycosylated protein with an apparent Mr of 42 kDa, is antigenic. In one experiment, this protein was shown to provide protection of piglets against PRRSV infection in the absence of a noticeable neutralizing humoral response. A minor fraction of GP ₃ is secreted (by transit through the secretory pathway) into cell culture fluids as a soluble protein (sGP ₃), whereas the bulk of the protein remains associated with the ER. Pigs exposed to the native form of GP ₅ by mean of DNA immunization, develop specific neutralizing and protecting antibodies. GP ₅ is also involved in antigenic variability, apoptosis and possibly, an antibody-dependent enhancement phenomena. GP ₄ also possesses antigenic determinants that trigger the immune system for the production of neutralizing antibodies. As for other enveloped RNA viruses, high degrees of genomic variabilities have been reported for arteriviruses, including PRRSV. Since the ORFs 2 to 7 protein sequences of European and North American PRRSV isolates differ by 21 to 45%, the isolates from these two continents are in fact two distinct viral species that must have diverged from a common ancestor. Each of the PRRSV structural proteins carries common and type-specific antigenic determinants that permit the ability to differentiate between European and North American strains. Live attenuated and killed vaccines are commercially available to protect pigs against PRRSV infection. In comparison to the killed vaccines, the attenuated vaccines induce an immunity against disease which lasts longer and is more efficient. However, these live vaccines can still be improved since they do not prevent reinfection. Genetically engineered vaccines can easily be designed to discriminate between vaccinated and naturally-infected pigs. However, to elicit a protective response, researchers would have to target antigens which are not only involved in the production of virus neutralizing antibodies, but also those antigens that are involved in the induction of an effective CMI response. The role of mucosal immunity should not be neglected, as it may be largely involved in the establishment of a protection against opportunistic infections that are favored following early pulmonary alveolar macrophages infection by PRRSV.