NLRP3 inflammasome activation by Foot-and-mouth disease virus infection mainly induced by viral RNA and non-structural protein 2B.

Foot-and-mouth disease virus (FMDV) is a positive-strand RNA virus of the family Picornaviridae. Early studies show that some viruses of Picornaviridae, such as EMCV and EV71, induce NLRP3 inflammasome activation. Our current study demonstrates that FMDV induces the secretion of caspase-1 and interleukin 1 beta (IL-1ß), as well as activates the NLRP3 inflammasome in a dose- and time-dependent manner. Meanwhile, NLRP3 inflammasome can suppress FMDV replication during virus infection. Both FMDV RNA and viroporin 2B stimulate NLRP3 inflammasome activation. FMDV RNA triggers NLRP3 inflammasome through p-NF-κB/p65 pathway not dependent on RIG-I inflammasome. FMDV 2B activates NLRP3 inflammasome through elevation of intracellular ion, but not dependent on mitochondrial reactive oxygen species (ROS) and lysosomal cathepsin B. It further demonstrates that 2B viroporin activates NLRP3 inflammasome and induces IL-1ß in mice, which enhances the specific immune response against FMDV as an ideal self-adjuvant for FMD VLPs vaccine in guinea pigs. The results reveal a series of regulations between NLRP3 inflammasome complex and FMDV. Amino acids 140-145 of 2B is essential for forming an ion channel. By mutating the amino acid and changing the hydrophobic properties, the helical transmembrane region of the viroporin 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.

Foot-and-mouth disease virus infection stimulates innate immune signaling in the mouse macrophage RAW 264.7 cells.

The innate immune system acts as the first line of defense against invasion by bacterial and viral pathogens. The role of macrophages in innate immune responses to foot-and-mouth disease virus (FMDV) is poorly understood. To determine the mechanism underlying activation of innate immunity after FMDV infection in macrophages, we performed FMDV infection in mouse macrophage RAW 264.7 cells and found that FMDV serotype O infection induced a cytopathic effect. We then evaluated the gene expression profile in macrophage RAW 264.7 cells after FMDV infection using systematic microarray analysis. Gene ontology annotation and enrichment analysis revealed that FMDV promoted expression in a group of genes that are enriched in innate immune response and inflammatory response processes. Further research demonstrated that FMDV serotype O infection enhanced NF-κB, Toll-like, and RIG-I-like receptor signaling pathways and proteins expression and increased transcription and expression of a series of cytokines and interferons, as proved by qRT-PCR, Western blot, ELISA, and dual-luciferase reporter assay. Our study concluded that FMDV infection triggers the innate immune response in macrophages after activation of multiple innate immune pathway receptors and proteins by FMDV serotype O, resulting in activation and secretion of a series of cytokines and interferons.

[Diversity of receptor recognition site of type Asial foot-and-mouth disease virus].

OBJECTIVE: Foot-and-Mouth Disease Virus (FMDV) initiates infection by binding to integrin receptors via a highly conserved Arg-Gly-Asp (RGD) sequence found in the G-H loop of the structural protein VP1. However, FMDV is an RNA virus, which harbors the evolutionary potential to render the RGD motif dispensable upon changes in constant environment. We studied mutation of RGD motif upon short-time passages FMDV Asial/JS/China/2005 field strain in different host. METHODS: VP1 gene was amplified from Asial/JS/China/05 field strain, the fourth passage virus in sulking mice (MF4) of the above strain and the virus isolated from a pig housing with cattle inoculated with the above strain followed eight passages in BHK-21 cell (PBF8) by RT-PCR, and the VP1 genes were sequenced and their deduced amino acid sequences were compared with each other. RESULT: Dominant population with RGD and Arg-Ser-Asp (RSD) receptor recognition site motif was generated after four passages of Asial/JS/China/2005 field virus in sulking mice and another dominant population with Arg-Asp-Asp (RDD) motif was produced after eight passages the virus isolated from housing pig with cattle inoculated with the above field strain. CONCLUSION: This study indicated that the dominant FMDV virus populations with RSD or RDD receptor binding site instead of original RGD motif were produced upon short-time evolution of FMDV field isolates with RGD motif in different environment. These studies not only increase number of viable mutants with substitutions in the RGD region, but also these profoundly altered, but viable, mutants with different receptor recognition site will provide useful tools for studies of cell recognition by FMDV and host tropism modifications.

Basal Level p53 Suppresses Antiviral Immunity against Foot-and-Mouth Disease Virus.

Tumor suppressor protein p53 (p53) is a master transcription factor that plays key roles in cell cycle arrest, apoptosis, senescence, and metabolism, as well as regulation of innate immunity during virus infection. In order to facilitate their replication and spreading, viruses have evolved to manipulate p53 function through different strategies, with some requiring active p53 while others demand reduction/inhibition of p53 activity. However, there are no clear-cut reports about the roles of p53 during the infection of foot-and-mouth disease virus (FMDV), the causative agent of a highly contagious foot-and-mouth disease (FMD) of cloven-hoofed animals. Here we showed that p53 level was dynamically regulated during FMDV infection, being degraded at the early infection stage but recovered to the basal level at the late stage. Cells depleted of p53 showed inhibited FMDV replication and enhanced expression of the immune-related genes, whereas overexpression of p53 didn't affect the viral replication. Viral challenge assay with p53 knockout mice obtained similar results, with viral load decreased, histopathological changes alleviated, and lifespan extended in the p53 knockout mice. Together, these data demonstrate that basal level p53 is required for efficient FMDV replication by suppressing the innate immunity.

Transcript Profiling Identifies Early Response Genes against FMDV Infection in PK-15 Cells.

Foot-and-mouth disease (FMD) is a highly contagious disease that results in enormous economic loses worldwide. Although the protection provided by vaccination is limited during early infection, it is recognized as the best method to prevent FMD outbreaks. Furthermore, the mechanism of host early responses against foot-and-mouth disease virus (FMDV) infection remains unclear. In our study, a pig kidney cell line (PK-15) was used as a cell model to reveal the mechanism of early pig responses to FMDV infection. Four non-treated control and four FMDV-treated PK-15 cells were sequenced with RNA-seq technology, and the differentially expressed genes (DEGs) were analyzed. The results showed that 1212 DEGs were in the FMDV-infected PK-15 cells, including 914 up-regulated and 298 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were significantly enriched in the tumor necrosis factor (TNF), cytokine-cytokine receptor interaction, NOD-like receptor, toll-like receptor, NF-κB, and the chemokine signaling pathways. To verify the results of the DEGs, 30 immune-related DEGs (19 up-regulated and 11 down-regulated) were selected for Quantitative Reverse Transcriptase polymerase chain reaction (RT-qPCR) verification. The results showed that RT-qPCR-measured genes exhibited a similar pattern as the RNA-seq analyses. Based on bioinformatics analysis, during FMDV early infection, we found that a series of cytokines, such as interleukins (IL6), chemokines (CXCL2, CCL20 and CCL4), and transcription factors (ZFP36, FOS, NFKBIA, ZBTB3, ZNF503, ZNF283, dymeclin (DYM), and orthodenticle homeobox 1 (OTX1)) were involved in the battle between FMDV and the host. Combined with their features and functions, we propose inflammation as the main early mechanism by which the host responds to FMDV infection. These data provide an additional panel of candidate genes for deciphering the mechanisms of a host's early response against FMDV infection.

Polyelectrolyte-based electrochemiluminescence enhancement for Ru(bpy)3²âº loaded by SiO2 nanoparticle carrier and its high sensitive immunoassay.

In this paper the strong electrochemiluminescence (ECL) nanoparticles have been prepared based on the anionic polyelectrolyte sodium polyacrylate (PAA)-ECL enhancement for Ru(bpy)3(2+), which were loaded by the carrier of SiO2 nanoparticle. There were two kinds of Ru(bpy)3(2+) for the as-prepared nanoparticles, the doped one and the exchanged one. The former was loaded inside the ECL nanoparticles by doping, in a form of ion-pair macromolecules PAA-Ru(bpy)3(2+). The corresponding ECL was enhanced about 2 times owing to the doping increase of Ru(bpy)3(2+). The latter was loaded on the PAA-doped Nafion membrane by ion exchange. The corresponding ECL was enhanced about 3 times owing to the ion-exchanging increase of Ru(bpy)3(2+). At the same time, ECL intensity of the doped-inside Ru(bpy)3(2+) was further enhanced 13 times because polyelectrolyte PAA in the doped membrane could obviously enhance electron transfer between the doped Ru(bpy)3(2+) and the working electrode. Furthermore, based on hydrophobic regions of the doped membrane antibody labeling could be easily realized by the as-prepared nanoparticles and then a high sensitive ECL immunoassay for HBsAg was developed. The linear range was between 1.0 and 100 pg mL(-1) (R(2)=0.9912). The detection limit could be as low as 0.11 pg mL(-1) (signal-to-noise ratio=3).

[Assessment research on the inter-proximal mesial papilla height of single implant-supported maxillary central incisor].

OBJECTIVE: This prospective study was to observe the correlation between the mesial papilla's height of single implant-supported maxillary central incisor and the distance from the base of the contact point to the alveolar bone crest. METHODS: 56 patients involved in single implant-supported maxillary central incisor were included in this study. The distances from the base of the contact point to the alveolar bone crest in the digital periapical film of maxillary central incisor were measured using the software Planmeca Dimaxis Version 3.3.2. The time of measurements were as follows: The pre-surgical and post-surgical periods, before and after the crown installation, the follow-up examination of more than 0.5 year. To analyze the factor of influencing distance, and the correlation between the distance and the height of gingival papilla during the whole restored period. Correlation analysis between the distance and the height of gingival papilla during the whole restoration was done by the statistical software SPSS 12.0. RESULTS: The results demonstrated that the ratio of esthetic papilla can achieve 54.5% at the crown installation and 95.5% at the follow-up examination when the distance was between 3 mm and 5 mm. When the distance was between 5 mm and 6 mm, they dropped to 30.0% and 75.0%, respectively. However, when the distance increased to above 7 mm, the papilla could hardly be in an esthetic outcome. There was a significant change of the distance was found during the periods from the post-surgery to pre-restoration, and the scope of the changes was between -0.13 mm and 0.46 mm. A negative correlation was found between the distance and the index of papilla. The correlation coefficient r was -0.715 (P < 0.01). CONCLUSION: It is proposed that the pre-surgery distance of maxillary central incisor from the base of the contact point to the alveolar bone crest can be used as one of the important reference indexes to assess and predict the height conditions of gingival papilla.