|Han, Jun - UNIVERSITY OF MINNESOTA|
|Kehrli Jr, Marcus|
|Rutherford, Mark - UNIVERSITY OF MINNESOTA|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 14, 2008
Publication Date: June 22, 2008
Citation: Faaberg, K.S., Han, J., Lager, K.M., Kehrli, Jr., M.E., Rutherford, M.S. 2008. PRRSV Strain VR-2332 Nsp2 Deletion Mutants Attenuate Clinical Symptoms in Swine [abstract]. XIth International Symposium on Nidoviruses. Paper No. P63. p. 70. Technical Abstract: PRRSV nonstructural protein 2 (nsp2) contains a N-terminal cysteine proteinase (PL2) domain, a middle hypervariable region and C-terminal putative transmembrane domain. We initially investigated the proteolytic processing of nsp2 PL2 in infected MARC-145 cells. A c-myc epitope was inserted into a deletion mutant (delta324-434) of recombinant Type 2 PRRSV (rVR-V7) to facilitate nsp2 fragment identification. Using an antibody panel, 5 nsp2 processing products were revealed that corresponded to sizes with and without the predicted C-terminal transmembrane domain. Other deletion mutants were used to map positions of the cleavage sites, which were identified as aa 828, 981 and 1196. The 1196G|G|G cleavage site was critical to successful virus replication in cells. These results correlate with a previous study that showed that recombinants with hypervariable region deletions outside of amino acids 324-813 were non-viable. Prior studies had shown that as much as 403 amino acids could be removed from the hypervariable region without losing virus viability in vitro (Han et al., J. Virol, 2007). We utilized selected nsp2 deletion mutants to compare in vitro and in vivo growth. Young swine (4 pigs/group; 5 control swine) were inoculated intramuscularly with one of 4 nsp2 deletion mutants (delta727-813, delta543-726, delta324-523, delta324-726) and full-length recombinant virus (VR-V7). Serum samples were collected on various days post-inoculation. Samples were analyzed by HerdChek ELISA, RT-PCR, interferon gamma ELISA, and nucleotide sequence analysis. Lymph node weight compared to body weight was recorded for each animal and used as a clinical measurement of viral pathogenesis. Results showed that all deletion mutants grew less robustly than full-length recombinant virus, yet all but the large deletion virus (delta324-726) recovered to parental virus levels by study end. Swine receiving mutants delta727-813 and delta324-726 had a significant decrease in lymph node involvement compared to rVR-V7. Three of the 4 deletion mutants had significant reductions in serum IFN-gamma levels; only the '543-726 nsp2 mutant mimicked VR-V7 in inducing a host serum IFN-gamma response. Sequencing results suggested that all nsp2 deletions were stable. The data suggest that selected nsp2 deletion mutants may indicate regions responsible for inducing IFN-gamma and domains that cause lymph node enlargement, a significant advancement in our understanding of PRRSV pathogenesis.