Study On Neuropathogenesis Of Porcine Reproductive And Respiratory Syndrome Virus (PRRSV)

Porcine reproductive and respiratory syndrome (PRRS) characterized by severe reproductive failure in sows and respiratory disorders in piglets causes enormous economic losses to the swine industry. Since 2006, there have been devastating outbreaks of atypical porcine reproductive and respiratory syndrome in China, which is caused by a highly pathogenic PRRS virus (HP-PRRSV) with a 30-amino-acid (30 aa) deletion in non-structural protein 2 (NSP2). HP-PRRS is characterized by high fever, high morbidity, high mortality, and associated with severe neurological symptoms including shivering, lameness, opisthotonus etc., and causes nonsuppurative encephalitis in pigs. Virus infections of central nervous system (CNS) have potential destructiveness. Microglia are the resident innate immune cells in CNS, whose activation has been implicated as an important contributor to the pathogenesis of CNS diseases. However, whether PRRSV infects microglia and the neuropathogenesis of HP-PRRSV need more studies.In this study, we successfully cultured porcine microglia and demonstrated that microglia could support PRRSV infection and replication in vitro. We further showed that HP-PRRSV infection significantly up-regulated the key inflammatory factors including IL-1β, TNF-α, IL-6, IL-12, IL-8, CXCL10, MCP-1, CCL3, CCL4, and CCL5 in cultured microglia as well as in the CNS of HP-PRRSV-infected pigs. The transcription factors NF-κB and AP-1, which are widely reported to regulate cytokine and chemokine productions, were activated by HP-PRRSV infection in microglia. Meanwhile, we found that HP-PRRSV induced cellular ROS formation in microglia and ROS scavenger was proved to significantly abolish the activation of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-8), indicating that ROS are crucial for pro-inflammatory gene production. Importantly, incubation with supernatants from HP-PRRSV-infected microglia cell culture remarkably induced SH-SY5Y neuroblastoma cell death. Collectively, these results showed that PRRSV infection induced pro-inflammatory factors up-regulation in microglia, which might contribute to neurotoxicity.IL-1β is one of pro-inflammatory cytokines which has diverse functions in the immune response to infection, including activation of innate immune, and modulation of adaptive immune responses. In addition, considerable evidence indicates that IL-1β is involved in neuronal injury. In this study, we found that PRRSV infection up-regulated IL-1β expression at both the mRNA and protein levels in microglia. We subsequently demonstrated that MyD88, PKC, and ERK1/2 were critical in PRRSV-induced IL-1β production in microglia using MyD88 inhibitory peptide or specific inhibitors of PKC, PI3K, JNK, NF-κB and ERK MAPK. Then we showed that ERK1/2 was indeed phosphorylated and reached a peak at 12 hour post infection (hpi) in PRRSV-infected microglia by western blot analysis. Inflammsome plays an important role in IL-1β processing and secretion, and NLRP3 inflammasome has been shown to respond to diverse stimuli, including virus infection. To examine whether the NLRP3 inflammasome is involved in IL-1β secretion, NLRP3 expression was knockdown by siRNA or Caspase-1 was inhibited by Z-VAD-FMK. Our data showed that IL-1β secretion was significantly decreased, suggesting the importance of NLRP3 inflammasome in IL-1β production induced by HP-PRRSV.In summary, we demonstrated that cultured microglia in vitro supported PRRSV infection and replication. HP-PRRSV infection induced the expressions of many pro-inflammatory factor in microglia, which might contribute to neurotoxicity caused by HP-PRRSV. PRRSV infection could up-regulate IL-1β production in microglia, which is likely dependent on MyD88, PKC, and ERK1/2. NLPR3 inflammasome activation was also showed to be involved in IL-1β maturation induced by PRRSV. These data may help us understand the neuropathogenesis of HP-PRRSV infection in pigs.