Anning Li

Understanding PRRSV Infection in Porcine Lung Based on Genome-Wide Transcriptome Response Identified by Deep Sequencing

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illuminas digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.

Comparative Analyses by Sequencing of Transcriptomes during Skeletal Muscle Development between Pig Breeds Differing in Muscle Growth Rate and Fatness

Understanding the dynamics of muscle transcriptome during development and between breeds differing in muscle growth is necessary to uncover the complex mechanism underlying muscle development. Herein, we present the first transcriptome-wide longissimus dorsi muscle development research concerning Lantang (LT, obese) and Landrace (LR, lean) pig breeds during 10 time-points from 35 days-post-coitus (dpc) to 180 days-post-natum (dpn) using Solexa/Illuminas Genome Analyzer. The data demonstrated that myogenesis was almost completed before 77 dpc, but the muscle phenotypes were still changed from 77 dpc to 28 dpn. Comparative analysis of the two breeds suggested that myogenesis started earlier but progressed more slowly in LT than in LR, the stages ranging from 49 dpc to 77 dpc are critical for formation of different muscle phenotypes. 595 differentially expressed myogenesis genes were identified, and their roles in myogenesis were discussed. Furthermore, GSK3B, IKBKB, ACVR1, ITGA and STMN1 might contribute to later myogenesis and more muscle fibers in LR than LT. Some myogenesis inhibitors (ID1, ID2, CABIN1, MSTN, SMAD4, CTNNA1, NOTCH2, GPC3 and HMOX1) were higher expressed in LT than in LR, which might contribute to more slow muscle differentiation in LT than in LR. We also identified several genes which might contribute to intramuscular adipose differentiation. Most important, we further proposed a novel model in which MyoD and MEF2A controls the balance between intramuscular adipogenesis and myogenesis by regulating CEBP family; Myf5 and MEF2C are essential during the whole myogenesis process while MEF2D affects muscle growth and maturation. The MRFs and MEF2 families are also critical for the phenotypic differences between the two pig breeds. Overall, this study contributes to elucidating the mechanism underlying muscle development, which could provide valuable information for pig meat quality improvement. The raw data have been submitted to Gene Expression Omnibus (GEO) under series GSE25406.

Lipopolysaccharide-induced miR-1224 negatively regulates tumour necrosis factor-α gene expression by modulating Sp1.

The innate immune response provides the initial defence mechanism against infection by other organisms. However, an excessive immune response will cause damage to host tissues. In an attempt to identify microRNAs (miRNAs) that regulate the innate immune response in inflammation and homeostasis, we examined the differential expression of miRNAs using microarray analysis in the spleens of mice injected intraperitoneally with lipopolysaccharide (LPS) and saline, respectively. Following challenge, we observed 19 miRNAs up‐regulated (1·5‐fold) in response to LPS. Among these miRNAs, miR‐1224, whose expression level increased 5·7‐fold 6 hr after LPS injection and 2·3‐fold after 24 hr, was selected for further study. Tissue expression patterns showed that mouse miR‐1224 is highly expressed in mouse spleen, kidney and lung. Transfection of miR‐1224 mimics resulted in a decrease in basal tumour necrosis factor‐α (TNF‐α) promoter reporter gene activity and a down‐regulation of LPS‐induced TNF‐α mRNA in RAW264.7 cells. With public databases of miRNA target prediction, miR‐1224 was shown to bind to the 3′ untranslated region (UTR) of Sp1 mRNA, whose coding product controls TNF‐α expression at the transcriptional level. Furthermore, we found that in HEK‐293 cells, the activity of the luciferase reporter bearing Sp1 mRNA 3′ UTR was down‐regulated significantly when transfected with miR‐1224 mimics. After transfection of miR‐1224 in RAW264.7 cells, nucleus Sp1 protein level decreased, and when endogenous miR‐1224 was blocked, the decrease was abolished. Therefore, we initially speculated that miR‐1224 was a negative regulator of TNF‐α in an Sp1‐dependent manner, which was confirmed in vivo by chromatin immunoprecipitation assay, and might be involved in regulating the LPS‐mediated inflammatory responses.

Proteome changes of lungs artificially infected with H-PRRSV and N-PRRSV by two-dimensional fluorescence difference gel electrophoresis.

BackgroundPorcine reproductive and respiratory syndrome with PRRS virus (PRRSV) infection, which causes significant economic losses annually, is one of the most economically important diseases affecting swine industry worldwide. In 2006 and 2007, a large-scale outbreak of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) happened in China and Vietnam. However little data is available on global host response to PRRSV infection at the protein level, and similar approaches looking at mRNA is problematic since mRNA levels do not necessarily predict protein levels. In order to improve the knowledge of host response and viral pathogenesis of highly virulent Chinese-type PRRSV (H-PRRSV) and Non-high-pathogenic North American-type PRRSV strains (N-PRRSV), we analyzed the protein expression changes of H-PRRSV and N-PRRSV infected lungs compared with those of uninfected negative control, and identified a series of proteins related to host response and viral pathogenesis.ResultsAccording to differential proteomes of porcine lungs infected with H-PRRSV, N-PRRSV and uninfected negative control at different time points using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry identification, 45 differentially expressed proteins (DEPs) were identified. These proteins were mostly related to cytoskeleton, stress response and oxidation reduction or metabolism. In the protein interaction network constructed based on DEPs from lungs infected with H-PRRSV, HSPA8, ARHGAP29 and NDUFS1 belonged to the most central proteins, whereas DDAH2, HSPB1 and FLNA corresponded to the most central proteins in those of N-PRRSV infected.ConclusionsOur study is the first attempt to provide the complex picture of pulmonary protein expression during H-PRRSV and N-PRRSV infection under the in vivo environment using 2D-DIGE technology and bioinformatics tools, provides large scale valuable information for better understanding host proteins-virus interactions of these two PRRSV strains.

Characterization and Promoter Activity Analysis of a New Porcine Gene: NICE-3

Through comparative gene mapping, NICE-3, which is closely linked to tropomyosin 3 in human chromosome 1, was selected to be investigated as a new candidate gene associated with the muscle development in pigs. This gene was sequenced, chromosome mapped, expression analyzed, subcellularly localized, and promoter activity analyzed. After screening and sequencing, porcine NICE-3 was found in a bacterial artificial chromosome clone containing tropomyosin 3. Quantitative reverse transcription-polymerase chain reaction revealed that NICE-3 mRNA was widely expressed, with highest expression levels in longissimus dorsi muscles, followed by heart, biceps femoris, liver, kidney, back fat, and lowest expression levels in spleen, brain, lymph, lung, stomach, and small and large intestines. Fluorescence and confocal microscopy assay demonstrated that the fusion protein, GFP-NICE-3, was distributed throughout the cytoplasm, including the plasma membrane. NICE-3 was mapped to Sus scrofa chromosome 4, in a region of conserved synteny with human chromosome 1, where the homologous human gene is localized. Results of dual reporter gene assays and mutation experiments combined with electrophoresis mobility shift assays showed that the retinoid X receptor might be an important transcription factor affecting the promoter activity of this gene.