Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) has disrupted the global swine industry since the 1980s. PRRSV-host interactions are largely still unknown but may involve host ISG15 protein. In this study, we developed a monoclonal antibody (Mab-3D5E6) specific for swine ISG15 (sISG15) by immunizing mice with recombinant sISG15. A sandwich enzyme-linked immunosorbent assay (ELISA) incorporating this sISG15-specific Mab was developed to detect sISG15 and provided a lower limit of sISG15 detection of 200 pg/mL. ELISA results demonstrated that infection of porcine alveolar macrophages (PAMs) with low-virulence or attenuated PRRSV vaccine strains induced intracellular ISG15 expression that was independent of type I IFN production, while PAMs infection with a PRRSV vaccine strain promoted extracellular ISG15 secretion from infected PAMs. Conversely, the addition of recombinant sISG15 to PAMs mimicked natural extracellular ISG15 effects whereby sISG15 functioned as a cytokine by activating PAMs. Once activated, PAMs could inhibit PRRSV replication and resist infection with PRRSV vaccine strain TJM. In summary, a sandwich ELISA incorporating homemade anti-ISG15 Mab detected ISG15 secretion induced by PAMs infection with a PRRSV vaccine strain. Recombinant ISG15 added to cells exhibited cytokine-like activity that stimulated PAMs to assume an anti-viral state that enabled them to inhibit PRRSV replication and resist viral infection.
Highlights
Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense, single-stranded, enveloped RNA virus which belongs to the genus Porartevirus [1,2]
Since anti-swine Interferon-stimulated gen 15 (ISG15) (sISG15) monoclonal antibody (Mab)-3D5E6 was suitable for Immunofluorescence Assay (IFA), we proposed that it would be applied as the capture antibody for sandwich enzyme-linked immunosorbent assay (ELISA)-based detection of extracellular sISG15 from liquid samples
Mab-6D10 probing of the same Western Blot (WB) membranes, the results suggested that the PRRSV replication level was not directly related to the ISG15 protein level
Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense, single-stranded, enveloped RNA virus which belongs to the genus Porartevirus [1,2]. ISG15 could be secreted from monocytes and lymphocytes exhibited cytokine-like activities [21,22]; such activities included stimulation of NK cell activation, induction IFN-γ production of T-cell in a dose-dependent manner [21], and neutrophil chemotactic factor-like activity [22,23]. The cytokine-like role played by secreted ISG15 during the PRRSV infection has remained elusive, due to the unavailability of anti-sISG15 antibody. The addition of recombinant sISG15 to PAMs prior to inoculation with a virulent PRRSV strain appeared to induce an antiviral state in the PAMs and strongly inhibit viral replication in PAMs. Taken together, our data suggest that ISG15 may function as an antiviral cytokine in PAMs such that the induction of secreted (extracellular) sISG15 is related to the virulence phenotype of PRRSV strain in vivo
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