The enormous genetic and antigenic diversity of PRRSV has become a diagnostic concern as it interferes with the accuracy of diagnostic tests and hampers the development of effective vaccines and the eradication of the disease. This study was conducted to assess the effects of genetic variation on serologic diagnosis and cross protection by antibody among different PRRS viruses and to identify the genetic elements critically associated with cross protection. Identification of the important genetic elements for cross protection would be useful not only to classify the viruses according to their immunologic relatedness but also to develop better disease-control methods including vaccines.
Three independent studies were designed to accomplish the stated objectives. The first study was conducted to determine if serologic data and the performance of serologic assays could be influenced by genotypic and/or biotypic differences of PRRS viruses and, if so, to assess the degree of effect. In the study, a comparative serologic study was conducted on five field and two cell-attenuated viruses to determine if serologic responses to PRRS virus infection could be influenced by biotypic and/or genotypic differences of the viruses. The isolates used for the study varied in their virulence to pigs and in genomic sequences. Ten pigs were inoculated with each isolate (1 x 103 TCID50) via the intranasal route. All inoculated animals became viremic during the study period. Some animals inoculated with the attenuated viruses remained seronegative until the end of the study (42 days PI), but all of the animals inoculated with field viruses developed ELISA- and IFA-detectable antibodies, regardless of the virus strain used in the IFA assay. In contrast, a great degree of variation in the onset and level of serum virus neutralization (SVN) antibody was observed by individual pigs and by each virus. The reactivity of SVN antibody was highly specific for homologous viruses. Therefore, it was concluded that the biotypic differences among PRRS viruses may affect the kinetics of humoral immune response in infected pigs. In addition, the IFA test may be used as a confirmatory test when a false-positive ELISA result is suspected or vise-a-versa at least among North American strains (PRRS virus type 2), but SVN antibody titers are highly affected by antigenic variability.
The second study was to identify genetic determinants associated with cross protection in ORF5 that encodes the major envelop protein (GP5) since GP5 has been postulated to be the most important protein to induce SVN antibody. The genetic elements within ORF5 which affect cross-neutralization were determined by genetically comparing field isolates which were classified according to their relative susceptibility to SVN antibody raised against VR2332 strain (North American prototype PRRS virus). In addition, the mutants in which the amino acid sequences were substituted with those found in the viruses resistant to SVN antibody at specific sites in ORF5 were generated using a VR2332-backboned infectious cDNA clone and site mutagenesis to confirm the role of those identified sites. Five common sites/domains (I to V) were identified in ORF5 from the sequence comparison after sixty-nine field isolated were classified based on the result of in vitro SVN test and/or animal challenge after passive immunization of SVN antibody. This suggests that the changes in amino acid sequences at three sites (32-34, 38-39, and 57-59) located in the N-terminal ectodomain of ORF5 significantly affected the susceptibility of the viruses to SVN antibody.
Finally, the third study was performed to assess the role of other structural proteins besides GP5 in cross protection among PRRS viruses and to define the corresponding genetic elements in each protein. In this study, chimeric mutants were generated by replacing ORF5 of an infectious clone constructed based on VR-2332 sequences with that of JA142, SDSU73, PRRS124, or 2M11715 to assess the role of ORF5 in cross neutralization. These viruses were genetically and antigenically distinct from VR-2332. In addition, chimeric mutants were constructed by substituting single or multiple structural genes of the VR-2332-infectious clone with the corresponding gene(s) of JA142. Virus neutralization test was performed on all mutants to determine the affect of substitutions on the susceptibility or resistance of viruses to the neutralizing activity of antisera generated against VR-2332, JA142, SDSU73 and PRRS124. All ORF5-replaced mutants showed the level of susceptibility or resistance close to that of the donor strains against homologous or heterologouos antisera but failed to achieve a complete reversion of cross neutralization. In contrast, substitution of ORFs 3-6 completely reversed the susceptibility of viruses to the neutralizing activity of anti-VR-2332 or JA142 antiserum. ORFs 3, 5, and 6 were additively responsible for such reversion between VR-2332 and JA142. These results indicate that the genetic similarity of ORFs 3 and 6 besides ORF5 should be taken into consideration to achieve the full-capacity of virus neutralization between two different PRRS viruses.
In conclusion, genetic variation of PRRSV negatively impacts cross neutralization among PRRS viruses. The similarity of specific amino acid determinants in GP3, GP5 and M proteins may significantly contribute to the level of cross protection between two viruses.