Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2010
Publication Date: 12/1/2010
Citation: Faaberg, K.S., Kehrli, Jr., M.E., Lager, K.M., Guo, B., Han, J. 2010. In vivo growth of porcine reproductive and respiratory syndrome virus engineered Nsp2 deletion mutants. Virus Research. 154(1-2):77-85. Interpretive Summary: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine in the United States, yet there is scant information available about the regions of the virus, PRRSV, that are responsible for the swine symptoms seen. One area of interest is viral nonstructural protein 2 (nsp2) located in the replicase protein. Prior studies had shown that as much as 403 amino acids could be removed from a large middle hypervariable region without losing virus viability in vitro. We utilized selected nsp2 deletion mutants to test the in vivo growth and investigate regions that may be associated with disease. Results showed that all deletion mutants grew less robustly than full-length recombinant virus, yet all but a large deletion virus recovered to parental virus levels by study end. Swine receiving one mutant had a significant decrease in lymph node involvement compared to rVR-2332. Three of the 4 deletion mutants had significant reductions in serum IFN-gamma levels; only one different nsp2 mutant mimicked rVR-2332 in inducing a host serum IFN-gamma response but exhibited a two-week delay. Sequencing results showed that all nsp2 deletions were stable. The data suggested that the selected nsp2 deletion mutants may indicate regions responsible for inducing high levels of serum IFN-gamma, an innate cytokine of unknown function in PRRSV clearance, and domains that cause lymph node enlargement, and thus represents a significant advancement in our understanding of PRRSV pathogenesis.
Technical Abstract: Prior studies on PRRSV strain VR-2332 nonstructural protein 2 (nsp2) had shown that as much as 403 amino acids could be removed from the hypervariable region without losing virus viability in vitro. We utilized selected nsp2 deletion mutants to examine in vivo growth. Young swine (4 pigs/group; 5 control swine) were inoculated intramuscularly with one of 4 nsp2 deletion mutants (rDelta727-813, rDelta543-726, rDelta324-523, rDelta324-726) or full-length recombinant virus (rVR-2332). Serum samples were collected on various days post-inoculation and analyzed by HerdChek* ELISA, PRRSV RT-PCR, gamma interferon (IFN-gamma) ELISA, and nucleotide sequence analysis of the entire nsp2 coding region. Tracheobronchial 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 (rDelta324-726) recovered to parental viral RNA levels by study end. Swine receiving the rDelta727-813 mutants had a significant decrease in lymph node enlargement compared to rVR-2332. While swine infection with rVR-2332 caused a rapid rise in serum IFN-gamma levels, the IFN-gamma protein produced by infection with 3 of the 4 deletion mutant viruses was significantly reduced, perhaps due to differences in viral growth kinetics. The rDelta543-726 nsp2 mutant virus, although growth impaired, mimicked rVR-2332 in inducing a host serum IFN-gamma response but exhibited a two-week delay. Targeted sequencing showed that all deletions were stable in the region coding for nsp2 after one swine passage. The data suggested that the selected nsp2 deletion mutants were growth attenuated in swine, altered the induction of serum IFN-gamma, an innate cytokine of unknown function in PRRSV clearance, and pointed to a domain that may influence tracheobronchial lymph node size.