| The porcine reproductive and respiratory syndrome virus (PRRSV) non-structural proteins (nsps) are predicted to be cleaved into 12 polypeptides, including nsp 1α, nsp 1β, and nsp 2 to 12, which mediate transcription and genome replication of a nested set of subgenomic mRNAs that encode structural proteins of the virus. Very little information is known about the function and structure of PRRSV nsps. Basic knowledge and techniques to better understand the function of nsps of PRRSV has been gained through similar studies of the Equine Arteritis Virus (EAV) which is placed in the same family as PRRSV.;Monoclonal and polyclonal antibodies are two key reagents to better study protein structure-function and to better design antiviral intervention strategies. Therefore, the overall objective of this project was to generate a full panel of antibodies against PRRSV nsps. Recombinant proteins encoding the predicted functional domains of nsps 2, 4, 9, 10, and 11 and proteins encoding nsps 1, 7, 8, and 12 were cloned in a pET-24b vector and in vitro expressed in E. coli BL21(DE3) (provided by Dr. Michael Murtaugh, University of Minnesota). Also, synthetic peptides were synthesized and conjugated to KLH as immunogens for nsps 3, 5, 6, and 10. BALB/c mice were immunized with 50 μg of either recombinant or synthetic protein antigens at two-week intervals for eight weeks, next, B-cells (splenocytes) were fused with NS-1 myeloma cells. Direct ELISAs and indirect immunofluorescent assays (IFA) were used to screen for specific anti-PRRSV mAbs. New Zealand white rabbits were immunized with recombinant or synthetic proteins at two-week intervals for eight weeks and exsanguanated. Direct ELISAa and IFA were used to screen for polyclonal antibodies. Hybridoma clones secreting specific mAbs were initially screened against VR-2332 PRRSV. Hybridomas secreting PRRSV-specific mAbs were cloned and ascites fluid was produced in mice. The monoclonal antibodies were isotyped and further analyzed by Western blotting and IFA. A panel of mAbs against nsp 1, 2, 4, 7, and 8 and a panel of polyclonal antibodies against nsp 1, 7, 9 and 11 were produced. No antibodies are currently available for the remaining nsps including proteins 3, 5, 6, 10, and 12. The project described here will provide the basic reagents for the study of the fundamental biology of PRRSV nsps. Also, these antibodies will provide good candidates for the development of new diagnostic assays to detect and distinguish different PRRSV strains which will help eliminate the PRRSV from swine herds throughout North America. |
