Inhibition of highly pathogenic PRRSV replication in MARC-145 cells by artificial microRNAs

<p>Abstract</p> <p>Background</p> <p>Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) has caused large economic losses in swine industry in recent years. However, current antiviral strategy could not effectively prevent and control this disease. In this research, five artificial microRNAs (amiRNAs) respectively targeted towards ORF5 (amirGP5-243, -370) and ORF6 (amirM-82, -217,-263) were designed and incorporated into a miRNA-based vector that mimics the backbone of murine miR-155 and permits high expression of amiRNAs in a GFP fused form mediated by RNA Pol II promoter CMV.</p> <p>Results</p> <p>It was found that amirGP5-370 could effectively inhibit H-PRRSV replication. The amirM-263-M-263, which was a dual pre-amiRNA expression cassette where two amirM-263s were chained, showed stronger virus inhibitory effects than single amirM-263. H-PRRSV replication was inhibited up to 120 hours in the MARC-145 cells which were stably transduced by recombinant lentiviruses (Lenti-amirGP5-370, -amirM-263-M-263). Additionally, efficacious dose of amirGP5-370 and amirM-263 expression did not trigger the innate interferon response.</p> <p>Conclusions</p> <p>Our study is the first attempt to suppress H-PRRSV replication in MARC-145 cells through vector-based and lentiviral mediated amiRNAs targeting GP5 or M proteins coding sequences of PRRSV, which indicated that artificial microRNAs and recombinant lentiviruses might be applied to be a new potent anti-PRRSV strategy.</p

Inhibition of HSP90 attenuates porcine reproductive and respiratory syndrome virus production in vitro

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) infection leads to substantial economic losses to the swine industry worldwide. However, no effective countermeasures exist to combat this virus infection so far. The most common antiviral strategy relies on directly inhibiting viral proteins. However, this strategy invariably leads to the emergence of drug resistance due to the error-prone nature of viral ploymerase. Targeting cellular proteins required for viral infection for developing new generation of antivirals is gaining concern. Recently, heat shock protein 90 (HSP90) was found to be an important host factor for the replication of multiple viruses and the inhibition of HSP90 showed significant antiviral effects. It is thought that the inhibition of HSP90 could be a promising broad-range antiviral approach. However, the effects of HSP90 inhibition on PRRSV infection have not been evaluated. In the current research, we tried to inhibit HSP90 and test whether the inhibition affect PRRSV infection. METHODS: We inhibit the function of HSP90 with two inhibitors, geldanamycin (GA) and 17- allylamono-demethoxygeldanamycin (17-AAG), and down-regulated the expression of endogenous HSP90 with specific small-interfering RNAs (siRNAs). Cell viability was measured with alamarBlue. The protein level of viral N was determined by western blotting and indirect immunofluorescence (IFA). Besides, IFA was employed to examine the level of viral double-stranded RNA (dsRNA). The viral RNA copy number and the level of IFN-β mRNA were determined by quantitative real-time PCR (qRT-PCR). RESULTS: Our results indicated that both HSP90 inhibitors showed strong anti-PRRSV activity. They could reduce viral production by preventing the viral RNA synthesis. These inhibitory effects were not due to the activation of innate interferon response. In addition, we observed that individual knockdown targeting HSP90α or HSP90β did not show dramatic inhibitory effect. Combined knockdown of these two isoforms was required to reduce viral infection. CONCLUSIONS: Our results shed light on the possibility of developing potential therapeutics targeting HSP90 against PRRSV infection

Characterization, expression profiles, intracellular distribution and association analysis of porcine PNAS-4 gene with production traits

<p>Abstract</p> <p>Background</p> <p>In a previous screen to identify differentially expressed genes associated with embryonic development, the porcine <it>PNAS-4 </it>gene had been found. Considering differentially expressed genes in early stages of muscle development are potential candidate genes to improve meat quality and production efficiency, we determined how porcine <it>PNAS-4 </it>gene regulates meat production. Therefore, this gene has been sequenced, expression analyzed and associated with meat production traits.</p> <p>Results</p> <p>We cloned the full-length cDNA of porcine <it>PNAS-4 </it>gene encoding a protein of 194 amino acids which was expressed in the Golgi complex. This gene was mapped to chromosome 10, q11–16, in a region of conserved synteny with human chromosome 1 where the human homologous gene was localized. Real-time PCR revealed that <it>PNAS-4 </it>mRNA was widely expressed with highest expression levels in skeletal muscle followed by lymph, liver and other tissues, and showed a down-regulated expression pattern during prenatal development while a up-regulated expression pattern after weaning. Association analysis revealed that allele C of SNP A1813C was prevalent in Chinese indigenous breeds whereas A was dominant allele in Landrace and Large White, and the pigs with homozygous CC had a higher fat content than those of the pigs with other genotypes (<it>P </it>< 0.05).</p> <p>Conclusion</p> <p>Porcine <it>PNAS-4 </it>protein tagged with green fluorescent protein accumulated in the Golgi complex, and its mRNA showed a widespread expression across many tissues and organs in pigs. It may be an important factor affecting the meat production efficiency, because its down-regulated expression pattern during early embryogenesis suggests involvement in increase of muscle fiber number. In addition, the SNP A1813C associated with fat traits might be a genetic marker for molecular-assisted selection in animal breeding.</p

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

<p>Abstract</p> <p>Background</p> <p>Porcine 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.</p> <p>Results</p> <p>According 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.</p> <p>Conclusions</p> <p>Our 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.</p

Aberrant host immune response induced by highly virulent PRRSV identified by digital gene expression tag profiling

<p>Abstract</p> <p>Background</p> <p>There was a large scale outbreak of the highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China and Vietnam during 2006 and 2007 that resulted in unusually high morbidity and mortality among pigs of all ages. The mechanisms underlying the molecular pathogenesis of the highly virulent PRRS virus (H-PRRSV) remains unknown. Therefore, the relationship between pulmonary gene expression profiles after H-PRRSV infection and infection pathology were analyzed in this study using high-throughput deep sequencing and histopathology.</p> <p>Results</p> <p>H-PRRSV infection resulted in severe lung pathology. The results indicate that aberrant host innate immune responses to H-PRRSV and induction of an anti-apoptotic state could be responsible for the aggressive replication and dissemination of H-PRRSV. Prolific rapid replication of H-PRRSV could have triggered aberrant sustained expression of pro-inflammatory cytokines and chemokines leading to a markedly robust inflammatory response compounded by significant cell death and increased oxidative damage. The end result was severe tissue damage and high pathogenicity.</p> <p>Conclusions</p> <p>The systems analysis utilized in this study provides a comprehensive basis for better understanding the pathogenesis of H-PRRSV. Furthermore, it allows the genetic components involved in H-PRRSV resistance/susceptibility in swine populations to be identified.</p

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/Illumina's 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

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/Illumina's 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