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R. Rowland 2 and Frank Blecha 1 * Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome (PRRS) Viruses Expressing Indicator Proteins and Antiviral Cytokines 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 ada porcine arterivirus virus cDNA infectious clone indicator proteins type I interferon host factors tumor susceptibility gene 101 2012-01-18T08:12:37+01:00 PScript5.dll Version 5.2.2 2012-01-18T08:13:37+01:00 2012-01-18T08:13:37+01:00 Acrobat Distiller 10.1.2 (Windows) porcine arterivirus; virus cDNA infectious clone; indicator proteins; type I interferon; host factors; tumor susceptibility gene 101 uuid:f920cc80-9a5a-4187-ba5f-4c5fec636eec uuid:62c038fb-1c25-4812-bf79-a9c424e0a2d3 False endstream endobj 7 0 obj <> endobj 35 0 obj <> endobj 36 0 obj < IFN- > I\ FN3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thu\ s allowing high throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to cl\ early 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, wh\ ich are capable of reversing subverted innate immune responses and may induce more effective ada)/Title(Replication-Competent Recombinant Porcine Reproductive and Respiratory Syndrome \(PRRS\) Viruses Expressing Indicator Proteins and Antiviral Cytokines)>> endobj xref 0 1 0000000000 65535 f 4 1 0000093747 00000 n 7 1 0000100744 00000 n 35 2 0000100839 00000 n 0000100888 00000 n trailer <<32A92461B628F54DB569181EF39502D8>]/Prev 116>> startxref 105444 %%EOF