Porcine epidemic diarrhea virus (PEDV) is

Background and purpose: The inflammation-resolving lipid mediator resolvin E1 (RvE1) effectively stops inflammationinduced bone loss in vivo in experimental periodontitis. It was of interest to determine whether RvE1 has direct actions on osteoclast (OC) development and bone resorption. Experimental approach: Primary OC cultures derived from mouse bone marrow were treated with RvE1 and analysed for OC differentiation, cell survival and bone substrate resorption. Receptor binding was measured using radiolabelled RvE1. Nuclear factor (NF)-kB activation and Akt phosphorylation were determined with western blotting. Lipid mediator production was assessed with liquid chromatography tandem mass spectrometry. Key results: OC growth and resorption pit formation were markedly decreased in the presence of RvE1. OC differentiation was inhibited by RvE1 as demonstrated by decreased number of multinuclear OC, a delay in the time course of OC development and attenuation of receptor activator of NF-kB ligand-induced nuclear translocation of the p50 subunit of NF-kB. OC survival and apoptosis were not altered by RvE1. Messenger RNA for both receptors of RvE1, ChemR23 and BLT 1 is expressed in OC cultures. Leukotriene B 4 (LTB 4 ) competed with [ 3 H]RvE1 binding on OC cell membrane preparations, and the LTB 4 antagonist U75302 prevented RvE1 inhibition of OC growth, indicating that BLT 1 mediates RvE1 actions on OC. Primary OC synthesized the RvE1 precursor 18R-hydroxy-eicosapentaenoic acid and LTB 4 . Co-incubation of OC with peripheral blood neutrophils resulted in transcellular RvE1 biosynthesis. Conclusions and implications: These results indicate that RvE1 inhibits OC growth and bone resorption by interfering with OC differentiation. The bone-sparing actions of RvE1 are in addition to inflammation resolution, a direct action in bone remodelling.

The polyunsaturated ω-3 fatty acid EPA-derived resolvin E1 (RvE1) enhances resolution of inflammation, prevents bone loss and induces bone regeneration. While the inflammation-resolving actions of RvE1 are characterized, molecular mechanism of its bone-protective actions are of interest. To test the hypothesis that receptor-mediated events impact bone changes, we prepared transgenic mice over expressing the RvE1 receptor chemR23 on leukocytes. In zymosan-initiated peritonitis, neutrophil polymorphonuclear leukocyte infiltration in response to RvE1 was limited requiring log order lower doses in chemR23tg mice. Ligature-induced alveolar bone loss was diminished in chemR23tg mice. Local RvE1 treatment of uniform craniotomy in the parietal bone significantly accelerated regeneration of the bone defect. In in vitro bone cultures, RvE1 significantly enhanced expression of osteoprotegerin (OPG) without inducing change in RANKL levels, while the osteogenic markers alkaline phosphatase, bone sialoprotein and Runt-related transcription factor 2 (RunX2) remained unchanged. These results indicate that RvE1 modulates osteoclast differentiation and bone remodeling by direct actions on bone, rescuing OPG production and restoring a favorable RANKL/OPG ratio, in addition to known anti-inflammatory and pro-resolving actions.

The role of polymorphonuclear neutrophils (PMN) in mediating diabetic tissue damage to the periodontium was investigated in a novel model of chronic hyperglycemia, the Akita mouse. Induction of acute peritoneal inflammation in wild-type (WT) and Akita mice resulted in exaggerated IL-6 response in Akita mice (2.9-fold increase over WT values) and a markedly increased chemokine response (KC, 2.6-fold; MCP-1, 2.6-fold; and MIP-1α, 4.4-fold increase over WT values). Chemotaxis to both fMLP and WKYMVm was significantly reduced in isolated Akita PMN compared with WT PMN as measured in a Boyden chamber. Superoxide release in contrast was significantly increased in Akita PMN as measured with cytochrome c reduction. Bone marrow-derived Akita PMN showed partial translocation of p47phox to the cell membrane without external stimulation, suggesting premature assembly of the superoxide-producing NADPH oxidase in hyperglycemia. In vivo studies revealed that ligature-induced periodontal bone loss is significantly greater in Akita mice compared with WT. Moreover, intravital microscopy of gingival vessels showed that leukocyte rolling and attachment to the vascular endothelium is enhanced in periodontal vessels of Akita mice. These results indicate that chronic hyperglycemia predisposes to exaggerated inflammatory response and primes leukocytes for marginalization and superoxide production but not for transmigration. Thus, leukocyte defects in hyperglycemia may contribute to periodontal tissue damage by impairing the innate immune response to periodontal pathogens as well as by increasing free radical load in the gingival microvasculature.

Mounting evidence has demonstrated that NLRP3 inflammasome plays a prominent role in the pathogenesis and progression of ischemic stroke, which indicates the higher possibility to target NLRP3 inflammasome in future stroke therapy. However, many aspects of the biology of NLRP3 inflammasome to stroke are still not well defined or even completely unknown. As the mechanistic insight of the NLRP3 inflammasomes increases, opportunities to develop new therapeutic strategies for patients with ischemic stroke are expected to enhance proportionately.

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle ( Hepatitis C virus (HCV) is a major cause of liver disease. A total of 170 million individuals worldwide are estimated to be infected with HCV and are at risk of developing cirrhosis and hepatocellular carcinoma (32,33). Unfortunately, there is presently no effective HCV vaccine available, and current treatments are far from satisfactory (22, 28). The development of antiviral therapies and effective vaccines has been hampered greatly by the lack of a convenient small-animal model. Chimpanzees (Pan troglodytes) are the only nonhuman primate host serving as an HCV infection model. However, experiments using chimpanzees are both expensive and ethically problematic. To date, three small-animal models of HCV infection have been reported: the immunotolerized rat model, Trimera mouse model, and uPA/SCID mouse model (16,25,39). However, these models are difficult to prepare, and the abnormal immune status of each greatly limits their application.The tree shrew or tupaia (Tupaia belangeri) is a small, squirrel-like mammal that is closely related to primates (6). Since the 1980s, tupaias have been used as an animal model of various infectious agents and their associated diseases. Tupaia has been shown to be susceptible to a variety of human viruses, including herpes simplex virus and hepatitis B virus (HBV) (9,38). Early in 1998 it was reported that inoculation with HCV RNA-positive serum could lead to short-term viremia and the appearance of anti-HCV IgG in tupaia (40). Furthermore, primary tupaia hepatocytes (PTHs) can be infected in vitro with serum derived from chronic hepatitis C patients (2, 18, 21, 36), although it is not clear whether the viral RNA measured is due to de novo production and/or from the virus inoculum. Recently, independent observations showed that inoculating tupaia with hepatitis C patient serum or viral particles reconstituted from full-length HCV cDNA caused mild hepatitis and intermittent viremia (1). However, these reports do not firmly establish the tupaia as a reliable model of HCV replication and pathogenesis. Importantly, patient sera often exhibit very weak infectivity (15,36). Although these results show promise, additional work has to be conducted to evaluate the value of pursuing the tupaia system in HCV research.The e...

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. Previous studies have reported that PRRSV infection induced the production of IL-1β. However, the cellular sensors and signaling pathways involved in this process have not been elucidated yet. Here, we studied the mechanisms responsible for the production of IL-1β in response to highly pathogenic PRRSV. Upon PRRSV infection of primary porcine alveolar macrophages, both mRNA expression and secretion of IL-1β were significantly increased in a time- and dose-dependent manner. We also investigated the role of several pattern-recognition receptors and adaptor molecules in this response and showed that the TLR4/MyD88 pathway and its downstream signaling molecules, NF-κB, ERK1/2, and p38 MAPKs, were involved in IL-1β production during PRRSV infection. Treatment with specific inhibitors or siRNA knockdown assays demonstrated that components of the NLRP3 inflammasome were crucial for IL-1β secretion but not for IL-1β mRNA expression. Furthermore, TLR4/MyD88/NF-κB signaling pathway was involved in PRRSV-induced expression of NLRP3 inflammasome components. Together, our results deciphered the pathways leading from recognition of PRRSV to the production and release of IL-1β, providing a deeper knowledge of the mechanisms of PRRSV-induced inflammation responses.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse-and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon beta (IFN-␤) production by cleaving the NF-B essential modulator (NEMO) at a single site, glutamate 349 (E349). Here, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-␤ production by targeting NEMO for proteolytic cleavage and that the scission occurred at four sites: E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-␤ production, we serendipitously found that a NEMO fragment (residues 1 to 349) could activate IFN-␤ transcription more robustly than full-length NEMO, whereas all other NEMO cleavage products were abrogated for the IFN-␤-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate the IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival. IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CL pro ) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrates that both EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is essential for disruption of type I IFN production. Moreover, we reveal that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has been observed only with picornavirus 3C proteases (3C pro ) and coronavirus 3CL pro . In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.KEYWORDS 3C-like protease, equine arteritis virus, NF-B essential modulator, interferon beta, porcine reproductive and respiratory syndrome virus T he family Arteriviridae, which belongs to the order Nidovirales, consists of porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) of mice, simian hemorrhagic fever virus RESULTSEAV nsp4-mediated inhibition of IFN-␤ production ...