Synthetic Toll-like Receptor 7 Ligand Inhibits Porcine Reproductive And Respiratory Syndrome Virus Infection In Primary Porcine Alveolar Macrophages

Porcine reproductive and respiratory syndrome virus(PRRSV) is an important swine infectious virus and pig is its only natural host. PRRSV infection causes reproductive failure in sows and respiratory distress in piglets. The disease can cause a seriously economic loss in the global swine industry. PRRSV can lead immunosuppression and persistent infection in pigs that have likely secondary infections. PRRSV has a high rate of mutation and new strains frequently appear in the field every year, which lead to the great difficulties to the control and prevention. The commercial vaccines currently available cannot provide a sustainable protection against PRRSV infection. Antiviral therapeutics provide a pivotal tool for combating viral infections, especially for cases in which existing vaccines strains fail to match the circulating virus strains, such as PRRSV. Therefore, antiviral pharmacological intervention may be an alternative measure to control PRRSV infection.Toll-like receptors(TLRs) are important pattern-recognition receptors, which mainly express in many immune cells, such as dendritic cells and macrophages, located in endosomes of cytoplasm. TLR7 can be activated by specific ligands, followed by recruiting adaptor molecules in the downstream and initiating signal cascade, in which the transcription factor NF-κB and IFN regulatory factor 7(IRF7) can be activated, leading to the production of inflammatory cytokines and IFNs to participate in antiviral responses. Previously, we synthesized a novel hydrosoluble TLR7 agonist, named SZU101, which demonstrated excellent anti-tumor property through activating TLR7 signal pathway. The objective of this study was to examine the effect of SZU101 on inhibiting PRRSV infection as TLR7 agonist or the immune adjuvant. The results of this study were shown as follows:1. PAMs were incubated with different concentration of SZU101 and then inoculated with PRRSV, the replication of virus was analyzed with IFA and qPCR methods both at gene and protein levels. The results showed that SZU101 inhibited PRRSV replication in PAMs in a dose-dependent manner. The reduction of progeny virus titers in the supernatant further demonstrated the antiviral effect of SZU101. Moreover, SZU101 inhibited different PRRSV strains, including CH-1a, JXA1, GD-HD and VR2332, indicating that the anti-PRRSV effect of SZU101 was not virus strain-dependent.2. To detect the direct role of SZU101 on PRRSV, PRRSV was incubated with SZU101 prior to virus infection. The results showed that pretreatment of PRRSV with SZU101 did not impair the virus infectivity. To test the antiviral activity of the cell culture media obtained from SZU101 treated PAMs, Marc-145 cells were incubated with the media before virus inoculation, resulting in significant inhibition of the virus replication. These results suggested that SZU101 induces antiviral cytokines production in PAMs. Further qPCR results showed that treatment of PAMs with SZU101 up-regulated the expression of IFNs(IFN-β and IFN-γ) and inflammatory cytokines(IL-12p40, IL-1β, IL-6 and TNF-α) as well as the expression of IRF7 and IFN-stimulated genes(ISG15 and IFIT1).3.SZU101 could activate NF-κB and in turn inhibit PRRSV replication in a dose-dependent manner in PAMs as shown by the western blot analysis of pNF-κB p65 levels. Blocking the function of TLR7 with its inhibitor abolished the SZU101 effect on the activation of NF-κB and the inhibition of PRRSV as well as the induction of cytokines and IFN related genes, indicating that the functional role of SZU101 was strictly TLR7 dependent. Abolishing the NF-κB function with its specific inhibitor reversed SZU101 effect on the PRRSV suppression and cytokines as well as IFN related genes induction, indicating that the activation of NF-κB was requisite for the SZU101 in its function progress. These results demonstrated that SZU101’s antiviral effects are dependedat least in part on TLR7-NF-κB signaling pathway.4. Balb/c mice were immunized with PRRSV combined with SZU101, the enhancement effect of SZU101 on the immune response against PRRSV was detected with ELISA and lymphocyte proliferation assay. The results showed that SZU101 could enhance the specific humoral and cellular immune responses against PRRSV in mice, suggesting SZU101 can be used as a potential adjuvant for PRRSV vaccines.