Replication-competent recombinant porcine reproductive and respiratory syndrome (PRRS) viruses expressing indicator proteins and antiviral cytokines

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more effective adaptive immunity against PRRSV infection.

Keywords: host factors; indicator proteins; porcine arterivirus; tumor susceptibility gene 101; type I interferon; virus cDNA infectious clone.

Figure 1

Infectious Porcine reproductive and respiratory syndrome virus (PRRSV) cDNA clone as a vector for bioengineered expression of indicator proteins. (A) Schematic of an infectious PRRSV cDNA clone, pCMV-P129-GFP, as an expression cassette [29]. (B) Co-infection of GFP- and DsRed-viruses (2nd passage of the rescued viruses, P2) in MARC-145 cells. Viruses were demonstrated by fluorescence of the indicator proteins. (C) Infection of porcine myeloid-derived dendritic cells with the Renilla luciferase (Rluc) (P6). Viruses were demonstrated by immunostaining PRRSV N protein indirectly labeled with TRITC. (D) Propagation dynamics of engineered viruses, values in parentheses are titers (log^TCID50/mL) of P2 viruses.

Figure 2

Indicator protein-expressing PRRS viruses are effective tools for deciphering the role of host factors in PRRSV infection. (A) Producing cell clones of MARC-145 cells with the suppression of a cellular protein of tumor susceptibility gene 101 (TSG101). (B1–D4) Retarded replication of PRRSV in TSG101-suppressed cells (B1–D2) as shown with DsRed-PRRSV in comparison to control cells (B3–D4). Merged: merged with fluorescent and bright field. (E) Real-time monitoring retarded replication of PRRSV in TSG101‑suppressed cells with the luciferase activity of the recombinant Rluc-PRRSV. The significant suppression of PRRSV replication in early phase but not latter phase of infection is clearly shown with the real-time measure using the indicator protein-expressing viruses, which is critical for detecting the window of a host factor involved in PRRSV infection. * p < 0.05, n = 3, to normal or scramble shRNA transfected cells.

Figure 3

Infectious PRRSV cDNA clone as a vector for bioengineered expression of immune effectors. (A–E) MARC-145 cells were transfected with equal amounts of indicated infectious constructs, and immunostained for PRRSV at 4 d post-transfection with antibody against the PRRSV N protein indirectly labeled with TRITC; (F) GFP‑PRRSV immunostained as in E1 but shown here as green fluorescence of GFP; (G) Merged (E1) and (F) to show specificity of the immunostaining procedure; (H) Propagation rate of IFN-engineered viruses, values in parentheses are titers (log^TCID50/mL) of P2 viruses. * p < 0.05, n = 3, to IFNα1-PRRSV.

Figure 4

Interferon (IFN)-engineered viruses express active IFN peptides and induce anti-PRRSV activity in MARC-145 cells. (A–D) Counterstained PRRSV and IFN-α1 in IFN-α1-PRRSV transfected cells at 4 d post transfection; (E) IFN-engineered PRRSV showed suppression of co-transfected (GFP-PRRSV) or co-infected (SDSU28983, infected at 3 d post transfection) viruses. Total virus-infected cells were examined at 5 d post transfection to calculate suppression rate in comparison to controls. ** p < 0.01, * p < 0.05, n = 3, to cells only transfected with GFP-PRRSV.