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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICAL STUDIES OF PUTATIVE NONSTRUCTURAL PROTEIN 2 IN PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS Project Number: 3625-32000-088-31
Project Type: Specific Cooperative Agreement

Start Date: Jun 08, 2009
End Date: Dec 14, 2009

Objective:
The objective of this cooperative research project is to elucidate the role(s) of one nonstructural protein (nsp) in PRRSV pathogenesis. The hypothesis is that PRRSV nonstructural protein 2 (nsp2) is a multifunctional regulator, influencing both viral replication and the host immune response. The specific aim of this cooperative agreement is examine whether the porcine immune response will be improved by modification of nsp2 in recombinant PRRSV by swine inoculation and diagnostic analysis of serum and blood samples.

Approach:
1. Twenty 4-6 week-old pigs, 5 pigs/group, will be acclimated for 5 days and then inoculated intranasally with 10**4 TCID50/ml of virus (2 nsp2 mutant viruses at passage 3, wt VR-2332, and strict negative control). Control pigs will receive PBS diluent. Each group of animals will be held in individual containment rooms in isolation facilities at the SDSU ARW. 2. Pigs will be monitored daily for clinical signs and blood (5-10 ml) will be collected in duplicate (heparinized and siliconized blood- collecting tubes) from each pig on day 3 and then on a weekly basis for approximately 2 months while monitoring PRRSV-specific antibody responses by IDEXX HerdChek ELISA and virus neutralization by the FFN assay at SDSU ARW. Neutralizing antibody responses are expected to reach maximum levels by 60 dpi, although the mutant viruses are predicted to result in enhance neutralization dynamics. 3. Aliquots of all serum samples will be retained at -80C for additional future analysis at SDSU ARW and the rest of the blood will be forwarded by the cooperator to the National Animal Disease Center (NADC), USDA-ARS, for further studies. 4. The NADC laboratory will perform nucleic acid sequence analysis on the nsp2 mutants before and after swine challenge, perform PRRSV Real-Time RT-PCR to assess viral load at times post-infection, and complete viral plaque assays. 5. The cooperator's laboratory will monitor the protein expression patterns of Th1/Th2 cytokines in PBMCs and inflammatory cytokines in serum by ELISA assay, and the cell-mediated immune response will be determined using the PRRSV specific INF-gamma ELISPOT. 6. If any of the nsp2 mutant infected pigs develop high levels of neutralizing Abs, all pigs will be challenged at 60 d.p.i. by infection with a virulent PRRSV strain (MN184) in order to determine to what extent the pigs may be protected from superinfection with a genetically dissimilar NA strain.

Last Modified: 9/22/2014
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