In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants

Abstract

Prior studies on PRRSV strain VR-2332 non-structural 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 (rΔ727-813, rΔ543-726, rΔ324-523, rΔ324-726) or full-length recombinant virus (rVR-2332). Serum samples were collected on various days post-inoculation and analyzed by HerdChek* ELISA, PRRSV real time RT-PCR, gamma interferon (IFN-γ) 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 (rΔ324-726) recovered to parental viral RNA levels by study end. Swine receiving the rΔ727-813 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-γ levels, the IFN-γ protein produced by infection with 3 of the 4 deletion mutant viruses was significantly reduced, perhaps due to differences in viral growth kinetics. The rΔ543-726 nsp2 mutant virus, although growth impaired, mimicked rVR-2332 in inducing a host serum IFN-γ response but exhibited a 2-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-γ, an innate cytokine of unknown function in PRRSV clearance, and pointed to a domain that may influence tracheobronchial lymph node size.

Epidemiology and Evolutionary Characteristics of the Porcine Reproductive and Respiratory Syndrome Virus in China between 2006 and 2010

“…Interestingly, a similar deletion in Nsp2 was observed in isolates from Guizhou province in 2007 (GenBank accession numbers EU140613, EU140614, and EU140618), implying that these isolates may have been prevalent in pigs in China. However, it is possible that these isolates were never widespread because the large deletions may affect the ability of the virus to survive (12). In addition to the deletions mentioned above, other types of deletions, i.e., of 12 amino acids, 68 amino acids, 65 amino acids, 51 amino acids, and 131 amino acids, have been observed in other isolates in previous studies (14,16,19,29,45).…”

An infectious clone of the highly pathogenic porcine reproductive and respiratory syndrome virus: Topology of glycoprotein 3 (GP3) addressing the intrachain disulfide bonds

“…As reported, GP3 might function collectively by interacting with GP2a and GP4 in virion as multimeric complex [29,33,34]. It also has been reported in EAV, GP3-GP2b/GP4 complex is disulfide linked when the virions leave the cells [34,35].…”

“…Two mutations in GP3, S83C and S117C in GP3 were demonstrated to be account for the decrease and delay in our study. Such kinds of decrease and delay also have been observed in the partly deletion of nsp2 in PRRSV, which could be explained by the deletion in nsp2 interfering the function of nsp2 [17,33].…”

Effect of Nonstructural Protein 2 Hypervariable Regions in the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Marc-145 Cells

“…The Nsp2 HV region has a complicated role in viral replication. Some studies suggest that a deletion in part of the Nsp2 HV region inhibits viral replication [37,38] . In contrast, some deletions in this region make PRRSV replication more efficient [39,40] .…”