The porcine microRNA transcriptome response to porcine cytomegalovirus infection
Ontology highlight
ABSTRACT:
ORGANISM(S): Sus scrofa
SUBMITTER: Xiao Liu
PROVIDER: E-GEOD-76156 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Porcine cytomegalovirus (PCMV; genus Cytomegalovirus, subfamily Betaherpesvirinae, family Herpesviridae) is an immunosuppressive virus that mainly inhibits the immune function of T lymphocytes and macrophages, which has caused great distress to the farming industry. In this study, we obtained the miRNA expression profiles of PCMV-infected and control porcine macrophages, PCMV-infected and control porcine tissues via high-throughput sequencing. The comprehensive analysis of miRNA profiles showed that 306 miRNA database annotated and 295 novel pig-encoded miRNAs were detected. Gene Ontology (GO) analysis of the target genes of miRNAs in PCMV infected porcine macrophages showed that the differentially expressed miRNAs are mainly involved in immune and metabolic process. This is the first report of the miRNA transcriptome in PCMV infected porcine macrophages and PCMV infected tissues and the analysis of the miRNA regulatory mechanism during PCMV infection. Further research into the regulatory mechanisms of miRNAs during immunosuppressive viral infections will contribute to the treatment and prevention of immunosuppressive viruses.
2015-12-19 | GSE76156 | GEO
Project description:Porcine cytomegalovirus (PCMV) is a member of the genus Cytomegalovirus, subfamily Betaherpesvirinae, and family Herpesvirus. PCMV is a major immunosuppressive virus that mainly suppress the immune function of T lymphocytes and macrophages. PCMV is widely distributed all over the world, but there are not significantly different serotypes found. Moreover, the molecular mechanisms of host anti-PCMV infection and the molecular immunosuppressive mechanisms of PCMV is still not well charaterized. To understand the PCMV potential impact on the function of immune organs, we performed microarray assay to analyze the transcriptome of porcine immune organs after PCMV infection. We identified 5582 differential expression genes by PCMV infection in microarray. There are 2161 upregulated genes and 3421 down-regulated by PCMV infection genes compare to the uninfected control group. We confirmed 13 differentially expressed immune-related genes by quantitative real-time RT-PCR (qPCR). Gene ontology, gene interaction networks and KEGG pathway analysis uncovered the differentially expressed genes interaction regulatory network. These findings indicated that PCMV regulates multiple functional pathways, including immune system process, cellular process, metabolic process, networks of cytokine-cytokine receptor interaction, TGF-beta signaling pathway, lymphocytes receptor signaling pathway and TNF signaling pathway. Our study is the first comprehensive attempt to explore the host transcriptional response to PCMV infection in porcine immune systems. It provided new insights into the immunosuppressive molecular mechanisms and pathogenesis of PCMV. This previously unrecognized endogenous antiviral mechanism has implications for development of host-directed strategies to the prevention and treatment of immunosuppressive viral diseases. 2 samples were analysed. PCMV infected porcine immune organs; control porcine organs. piglets were divided into two groups of five pigs each, and maintained under controlled temperature and humidity. Each pig in the first group was inoculated with 5 ml of 109 PFU/ml PCMV SC strain by intramuscular and intranasal injection, and the other five pigs were injected with 5 ml of RPMI-1640 nutrient solution (Thermo Fisher Scientific, Waltham, UK) as the control. The sera, lymph nodes, spleens, and thymuses of the infected and control pigs were collected at 14 dpi
2014-07-12 | E-GEOD-59115 | biostudies-arrayexpress
Project description:Porcine cytomegalovirus (PCMV) is a member of the genus Cytomegalovirus, subfamily Betaherpesvirinae, and family Herpesvirus. PCMV is a major immunosuppressive virus that mainly suppress the immune function of T lymphocytes and macrophages. PCMV is widely distributed all over the world, but there are not significantly different serotypes found. Moreover, the molecular mechanisms of host anti-PCMV infection and the molecular immunosuppressive mechanisms of PCMV is still not well charaterized. To understand the PCMV potential impact on the function of immune organs, we performed microarray assay to analyze the transcriptome of porcine immune organs after PCMV infection. We identified 5582 differential expression genes by PCMV infection in microarray. There are 2161 upregulated genes and 3421 down-regulated by PCMV infection genes compare to the uninfected control group. We confirmed 13 differentially expressed immune-related genes by quantitative real-time RT-PCR (qPCR). Gene ontology, gene interaction networks and KEGG pathway analysis uncovered the differentially expressed genes interaction regulatory network. These findings indicated that PCMV regulates multiple functional pathways, including immune system process, cellular process, metabolic process, networks of cytokine-cytokine receptor interaction, TGF-beta signaling pathway, lymphocytes receptor signaling pathway and TNF signaling pathway. Our study is the first comprehensive attempt to explore the host transcriptional response to PCMV infection in porcine immune systems. It provided new insights into the immunosuppressive molecular mechanisms and pathogenesis of PCMV. This previously unrecognized endogenous antiviral mechanism has implications for development of host-directed strategies to the prevention and treatment of immunosuppressive viral diseases.
2014-07-12 | GSE59115 | GEO
Project description:Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs. Certain miRNAs were differentially regulated during TGEV infection, which confirmed the hypothesis that specific miRNAs play a regulatory role in virus-host interactions. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process. This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases. 2 ST(Porcine testicular cells) cell samples, ST: normal ST cell sample (contro sample), TGEV: TGEV (Transmissible gastroenteritis virus) infected ST cell samples
2015-01-08 | E-GEOD-64737 | biostudies-arrayexpress
Project description:By analyzing the changes of miRNAs of piglet PAMs 6h after PRRSV infection,we can find porcine miRNAs associated with viral infection and possible target sites in both PRRSV and pig genome and their roles in protection against viral infections. Then these targets will screened by bioinformatics methods to find the susceptibility genes, resistance genes and immune related genes, and formed molecular biology network of miRNAs at gene and protein level . PAM cells were isolated from 60d old healthy piglets and inoculated with HuN4,a strain of highly pathogenic porcine reproductive and respitory syndrome virus (HP-PRRSV) and were collected at 6h post-inoculation (pi). The uninfected cells served as mock-infected cells.The miRNA chip was according to the porcine miRNA sequences in Sanger miRBase Release 17.0 (http://www.sanger.ac.uk/Software/Rfam/mirna/)added with some miRNAs we found in reference. Microarray hybridization and data analysis helps to find the susceptibility genes, resistance genes and immune related genes, and formed molecular biology network of miRNAs at gene and protein level .
2013-11-01 | E-GEOD-52014 | biostudies-arrayexpress
Project description:RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4(+) T-cell line, Jurkat. Jurkat cells with stably integrated HTLV1-LTR were transfected with either pCMV-Tax or pCMV. Total miRNA was isolated, labeled and hybridized to the miRNA microarray as described by the manufacturer (Invitrogen). The hybridized slides were then analyzed on a GenePix scanner.
2012-08-08 | E-GEOD-39986 | biostudies-arrayexpress
Project description:Background While more than 700 microRNAs (miRNAs) are known in human, a comparably low number has been identified in swine. Because of the close phylogenetic distance to humans, pigs serve as a suitable model for studying e.g. intestinal development or disease. Recent studies indicate that miRNAs are key regulators of intestinal development and their aberrant expression leads to intestinal malignancy. Results Here, we present the identification of hundreds of apparently novel miRNAs in the porcine intestine. MiRNAs were first identified by means of deep sequencing followed by miRNA precursor prediction using the miRDeep algorithm as well as searching for conserved miRNAs. Second, the porcine miRNAome along the entire intestine (duodenum, proximal and distal jejunum, ileum, ascending and transverse colon) was unraveled using customized miRNA microarrays based on the identified sequences as well as known porcine and human ones. In total, the expression of 332 intestinal miRNAs was discovered, of which 201 represented assumed novel porcine miRNAs. The identified hairpin forming precursors were in part organized in genomic clusters, and most of the precursors were located on chromosomes 3 and 1, respectively. Hierarchical clustering of the expression data revealed subsets of miRNAs that are specific to distinct parts of the intestine pointing to their impact on cellular signaling networks. Conclusions In this study, we have applied a straight forward approach to decipher the porcine intestinal miRNAome for the first time in mammals using a piglet model. The high number of identified novel miRNAs in the porcine intestine points out their crucial role in intestinal function as shown by pathway analysis. On the other hand, the reported miRNAs may share orthologs in other mammals such as human still to be discovered. RNA isolation from porcine intestinal tissue Intestinal samples (~ 2 cm circle segments) were taken from duodenum, proximal and distal jejunum, ileum, ascending and transverse colon of four 31 days old healthy piglets (EUROC x Pietrain). The piglets were weaned at the age of 28 days. Samples were quick-frozen in liquid nitrogen and stored at -80 °C. In order to obtain representative measurements in each intestinal locus, three cross sections of approximately 2 mm out of the 2 cm segment of frozen intestine were examined. These 3 sections were pooled and total RNA was isolated from samples using an automated homogenizer (FastPrep Instrument, MP Biomedicals, Heidelberg, Germany) and the mirVana miRNA Isolation Kit (Applied Biosystems, Darmstadt, Germany), according to the manufacturer’s protocol. The RNA quality and quantity of all samples were proven using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kits (Agilent, Waldbronn, Germany) and the Nanodrop 1000 Spectrophotometer (Thermo Scientific, MA, USA). Microfluidic miRNA microarrays Microfluidic microarray experiments were performed using customized Geniom biochips (febit). Customized microarrays were synthesized with the Geniom One device (febit) applying febit’s standard shortmer kit for oligonucleotide synthesis. The light-activated in situ oligonucleotide synthesis using a digital micromirror device was performed within the Geniom One instrument on an activated three-dimensional reaction carrier consisting of a glass-silicon-glass sandwich (biochip). Using standard DNA synthesis reagents and 3’-phosphoramidites carrying 5’-photolabile protective group, oligonucleotides were synthesized in parallel in eight individually accessible microchannels (referred to as microarrays) of one biochip. Prior to synthesis, the glass surface was activated by coating with spacer to facilitate probe-target interaction and to avoid probe-probe interface [37]. Two different microarray designs were used. The first one included only the 399 miRDeep predicted mature miRNAs and their star sequences in seven replicates allowing highly accurate validation of the miRDeep prediction plus 16 spike-in controls. The second design consisted of 1117 features in triplicate, including the identified conserved porcine mature miRNA sequences (18 mapping to the porcine genome and another 124, which did not map), 74 known porcine and 885 human miRNAs from miRBase (13.0) and 16 spike-in controls. For microarray experiments, intestinal total RNA samples from different loci of four subjects were isolated independently and respective pools of total RNA samples were prepared. All pools underwent a second quality control to determine the quality and quantity using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kit according to the manufacturer’s instructions. For each microarray 250 ng of total RNA were suspended in 25 µl of febit’s proprietary miRNA hybridization buffer. The hybridization was performed automatically for 16h at 42°C using the Geniom RT-Analyzer (febit). After stringent washing a microfluidic-based extension assay was performed to label the miRNAs using biotinylated nucleotides. After washing, biotinylated nucleotides were detected by streptavidin-phycoerythrin and signal recognition and calculation were done automatically within milliseconds. The background of the chips was effectively corrected by global background subtraction and inter-array effects were corrected by variance stabilizing normalization.
2010-05-05 | E-GEOD-21628 | biostudies-arrayexpress
Project description:The host response to S.typhimurium infections in ileum was studied after infection of piglets. mRNA and miRNA expression studies were performed to identify interactions of miRNAs and regulated mRNAs in context of mammalian intestinal Salmonella infection using a piglet model. Intestinal samples (~ 2 cm circle segments) were taken at time points 3h, 3d and 28d post infection from ileum of ten S.typhimurium infected piglets (S), ten S.typhimurium infected piglets and additional treated with probiotic strain E.faecium NCIMB 10415 (SP) and five healthy control piglets(C) (EUROC x Pietrain). The piglets were weaned at the age of 28 days and infected with S. typhimurium. Samples were quick-frozen in liquid nitrogen and stored at -80 M-BM-0C. In order to obtain representative measurements in each intestinal locus, three cross sections of approximately 2 mm out of the 2 cm segment of frozen intestine were examined. These 3 sections were pooled and total RNA was isolated from samples using an automated homogenizer (FastPrep Instrument, MP Biomedicals, Heidelberg, Germany) and the mirVana miRNA Isolation Kit (Applied Biosystems, Darmstadt, Germany), according to the manufacturerM-bM-^@M-^Ys protocol. The RNA quality and quantity of all samples were proven using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Kits (Agilent, Waldbronn, Germany) and the Nanodrop 1000 Spectrophotometer (Thermo Scientific, MA, USA). For microarray analysis pooled samples of each experimental group were prepared ana partes aequalis from individually isolated total RNA samples, which werde collected at the same time point in each group. Common reference was designed from equal amounts of total RNA of each pool and additional total RNA from ascending colon.
2013-06-25 | E-GEOD-40803 | biostudies-arrayexpress
Project description:Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is the most economically important disease in pig populations. Lung damage is one major pathological condition following PRRSV infection, often leading to animal death. In vivo, PRRSV productive infection occurs predominately in alveolar macrophages of the lung. Here, transcriptome profiling of pulmonary alveolar macrophages (PAMs) from Tongcheng piglets pre- and post- infection of highly pathogenic PRRSV has been performed using porcine Affymetrix GeneChip. All animal procedures were performed according to protocols approved by the Biological Studies Animal Care and Use Committee of Hubei Province, China. Piglets used in this study were free from PRRSV, pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2) determined by ELISA test for serum antibodies. Twelve of 5-week-old boars were obtained from three litters (four piglets per litter), and raised in pathogen-free facilities. In order to perform a paired experiment, every four full-sib individuals were divided equally into two groups: one infected group and one control group with 6 piglets in each group. The infected groups were challenged with PRRSV-Wuh2 (3 ml/15 kg, 10-5 TCID50/ml) by intramuscular inoculation. Slaughters were carried out at 0 days post-infection (dpi) for uninfected (control) groups, and at 5 or 7 dpi for infected groups. The PAMs for microarray analysis were collected by bronchoalveolar lavage from three uninfected pigs and three infected pigs at 5 dpi. Total of 6 microarrays have been hybridized in this experiment.
2012-06-30 | E-GEOD-22782 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE14758: Expression data from mediastinal lymph nodes of piglets experimentally infected with porcine circovirus type 2 (PCV2) GSE14790: Expression data from blood samples of piglets experimentally infected with porcine circovirus type 2 (PCV2) Refer to individual Series
2009-12-30 | E-GEOD-14791 | biostudies-arrayexpress