TCID50 analysis of FMDV titres in the supernatants. 2B is altered, so that the 2B is insufficient to trigger the activation of NLRP3 inflammasome. This study demonstrates the functions of FMDV RNA and 2B viroporin activate NLRP3 inflammasome and provides some useful information for the development of FMD vaccine self-adjuvant, which is also helpful for the establishment of effective prevention strategies by targeting NLRP3 inflammasome. strong class=”kwd-title” KEYWORDS: FMDV, FMDV RNA, viroporin, 2B, NLRP3 inflammasome, IL-1 1.?Introduction Foot-and-mouth disease (FMD) is an acute, febrile and contact infectious disease caused by Foot-and-mouth disease virus (FMDV) infection in cloven-hoofed animals. Therefore, after each outbreak, infected animals will be slaughtered and burned, which the production of livestock will decline markedly. In addition, there are individual variations of FMDV that can be transmitted to humans. FMD is known as the number one killer of animal husbandry. However, the molecular mechanisms by which FMDV stimulates inflammation are not defined. Macrophages play a pivotal role in triggering inflammation during FMDV infection . The innate immune system is a universal form of first-line defence of the host against virus invasions [2C5]. Its recognition relies on germline-encoded pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), retinoic acid-inducible gene (RIG-I)-like receptors, NOD-like INCB053914 phosphate receptors (NLRs), and C-type lectin receptors (CLRs) [6C14]. NLRs, as one of PRRs, recognize PAMPs (pathogen-associated molecular patterns) or DAMPs (damage-associated molecular patterns) and trigger assembly of inflammasome, which can lead to the activation of caspase-1 and the secretion of pro-inflammatory cytokines, such as IL-1 and IL-18, to further regulate the innate immune response . The NLRP3 inflammasome, including NLRP3, apoptosis-associated speck-like protein (ASC), and pro-Caspase-1, are the most fully identified. Once NLRP3 recognizes some signals of PAMPs or DAMPs, it oligomerizes through the oligomerization domain and directly recruits the ASC CARD domain through the N-terminal PYRIN domain of NLRP3. Afterwards, the CARD domain of ASC directly recruits pro-caspase-1 which will cleave to mature caspase-1 and finally make pro-IL-1 to IL-1, inducing the inflammation. HCV is a single-stranded positive-sense RNA virus, induces chronic inflammation and mediates liver damage. HCV induces IL-1/IL-18 mRNA expression through NF-B activation and produces IL-1 and IL-18 from a monocyte macrophage . HCV RNA can activate NLRP3 inflammasome and induce IL-1 secretion with transfection into monocytes or macrophages. HCV RNA activates signal 2 CD163 and triggers ASC oligomerization and caspase-1 cleavage not dependent on RIG-I. HCV RNA induces activation of the NLRP3 inflammasome dependent on ROS production . HCV RNA triggers NLRP3 inflammasome activation INCB053914 phosphate through MyD88-mediated TLR7 signalling to induce IL-1 mRNA expression and drive IL-1 secretion . Meanwhile, FMDV RNA is a single-stranded positive-sense RNA, which may be similar to HCV RNA. Many kinds of viruses can induce inflammatory responses and produce pro-inflammatory cytokines to stimulate NLRP3 INCB053914 phosphate inflammasome and prevent pathogenic invasions [19,20]. FMDV has not been reported whether it can also activate NLRP3 inflammasome. At present, some studies have reported FMDV can elicit an innate immune response in macrophage cells . Especially, Protein VP1, Lpro, 3Cpro, 2C, and 2B of FMDV can inhibit the expression of IFN-/, in which the cellular proteins, eIF4G, NEMO, KPNA1, and RIG-I are involved [21C28]. Previous studies confirm that FMDV 2B protein is a viroporin-like protein and forms hydrophilic pore at the cellular membranes [29C31]. Recently, some reports have shown that viroporins play an important role in the activation of the NLRP3 inflammasome by regulating the antiviral innate immune responses . These viroporins mainly include the influenza virus M2 protein [33C35], encephalomyocarditis virus (EMCV) 2B protein , human respiratory syncytial virus SH , human rhinovirus 2B protein , and hepatitis C virus (HCV) p7 protein [16,18]. Hence, it will be very interesting to find a new function of FMDV 2B in activation of the NLRP3 inflammasome. To assess the contribution of NLRP3 inflammasome and IL-1 secretion in FMDV pathogenesis, we firstly investigated the inflammasome response to FMDV infection and then focused on the FMDV RNA and 2B viroporin function of inflammasome activation. Our study reveals that FMDV can activate the NLRP3 inflammasome. NLRP3 inflammasome plays a protective role against FMDV infection. Furthermore, we find that FMDV RNA can activate p-NF-B/p65 pathway and viroporin 2B protein can induce ion efflux and trigger.