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.

Fig. 1

Genome and PRRSV deletion mutant schematic. Illustration of treatment groups 1–5 used for deletion mutant growth analyses and pathogenesis assessment. Downward arrows indicate predicted cleavage sites processed by PRRSV encoded PCPα and PCPβ (papain-like cysteine protease α and β; ▾) chymotrypsin-like cysteine protease (PL2; ) and serine/3C-like protease (3CLpro; ▿). Other signature motifs are also indicated: POL, RNA dependent RNA polymerase; C/H, cysteine/histidine rich; HEL, helicase; XendoU, Xenopus laevis homolog poly(U)-specific endoribonuclease. The short black bars () indicate predicted nsp2 transmembrane regions. Hypervariable regions of nsp2 (HV1 and HV2), defined previously, are shown in solid grey (Han et al., 2006).

Fig. 2

HerdChek* ELISA, detecting antibodies elicited against PRRSV nucleocapsid protein, were determined for all serum samples and then plotted as the mean and standard error of the mean for each group of pigs at all time points. Serum samples ≥0.4 (indicated by dotted line) were considered positive, as per manufacturer's guidelines (IDEXX).

Fig. 3

Virus titer results (TCID₅₀/ml) on all virus isolation positive serum samples. Virus isolation was performed with all serum samples and lavage fluids. Positive samples were then titered on fresh MARC-145 cells.

Fig. 4

Real-Time RT-PCR results, performed in duplicate, for all serum samples. Quantitative RT-PCR was used to detect, in duplicate, the PRRSV RNA copy number in 8 μl of viral RNA purified from serum.

Fig. 5

(A) RT-PCR detection of PRRSV construct used to infect 2 pigs/group (using days 4 and 7 viral RNA from serum samples; day 7 shown) in Group 1 – 808 bp product, Group 2 – 936 bp product, Group 3 – 608 bp product, Group 4 – 904 bp product, Group 5 – 674 bp product, and NC Group – no product expected. RT-PCR to confirm absense of rVR-2332 virus contamination in Group 1 – 592 bp product, Group 2 – 517 bp product, Group 3 – 538 bp product, and Group 4 – 955 bp product. Included were identical RT-PCR conditions for two pigs from Group 5 – 538 bp product (positive control) and NC group – no product expected. (B) Nested PCR was completed on Group 4 at time points that viral RNA was detected by Real-Time RT-PCR (492 bp product).

Fig. 6

Interferon γ protein ELISA for all serum samples were analyzed using a mixed linear model for repeated measures.