Project description:Porcine reproductive and respiratory disease (PRRS) is the most important disease in swine industry worldwide. However, strategies such as vaccination and good biosecurity are not consistently successful to eliminate PRRSV. Although some gene expression pathways have been explored recently, host molecular pathways blocked by PRRSV and the protective immune response expressed in pigs resistant to PRRSV are largely unknown. In order to answer these questions, we herein characterize changes in blood gene expression in pigs responding differentially to infection with a well characterized type 2 (North American) PRRSV isolate. Samples are those collected through the PRRS Host Genetics Consortium (PHGC). Samples were those from Tempus tube collected blood of PHGC pigs selected from four response groups according to their serum viral load (0-21 days post infection) and weight gain (0-42 dpi) and characterized as low vs. high viral load and low vs high weight gain .
Project description:Investigation of whole genome gene expression level changes in porcine alveolar macrophage response to co-infection of PRRSV and M. hyopneumoniae The results in this study will be further described in Bin li et al., 2013. BMC genomics. A 24 chip study using total RNA recovered from four separate group include control, PRRSV, M. hyopneumoniae and PRRSV+M. hyopneumoniae.
Project description:Porcine reproductive and respiratory disease (PRRS) is the most important disease in swine industry worldwide. However, strategies such as vaccination and good biosecurity are not consistently successful to eliminate PRRSV. Although some gene expression pathways have been explored recently, host molecular pathways blocked by PRRSV and the protective immune response expressed in pigs resistant to PRRSV are largely unknown. In order to answer these questions, we herein characterize changes in blood gene expression in pigs responding differentially to infection with a well characterized type 2 (North American) PRRSV isolate. Samples are those collected through the PRRS Host Genetics Consortium (PHGC). Samples were those from Tempus tube collected blood of PHGC pigs selected from four response groups according to their serum viral load (0-21 days post infection) and weight gain (0-42 dpi) and characterized as low vs. high viral load and low vs high weight gain . block reference design was used to accommodate samples from 4 treatment groups.
Project description:Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel virus of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of host genomic DNA (hgDNA) contamination. Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA but are cell membrane-impermeable and thus are unable to bind protected DNA and RNA such as viral genomic material. DNA modified by EMA or PMA is not amplifiable by polymerase. In order to assess the capacity of EMA or PMA to lower hgDNA, serum or lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with different combination of treatment: with or without ultracentrifugation and incubation with or without different concentration of EMA or PMA. PVDA and HTS were used to evaluate the capacity of both techniques to detect the presence of PRRSV from each sample. Negative results were obtained by PVDA and low amount of PRRSV specific reads were obtained by HTS with untreated samples or samples treated only by ultracentrifugation. An increase capacity of PRRSV detection was observable by PVDA in EMA and PMA treated samples but PVDA best results were obtained following PMA treatment, with or without ultracentrifugation. HTS sensitivity was also improved by a treatment with EMA or PMA, but the number of reads was significantly higher in PMA treated samples. These results support the use of PMA as a treatment to increase sensitivity of PVDA and HTS. A non specific DNA probe is used as a negative hybridization control. Also, specifics probes targeting pUC19 plasmid DNA is used as a DNA array positive control as well as a localization control. Finally, there are 34 probes of 70 nucleotides spotted in duplicate were selected to target PRRSV conserved regions. A total of 80 different tests were done by DNA array in order to evaluate different treatments conditions with EMA or PMA, with or without ultracentrifugation. A total of 58 samples were tested on specific lung tissue homogenates spiked with different concentration of PRRSV and 12 samples were serum samples spiked with one concentration of PRRSV. Finally. A total of six samples
Project description:Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel virus of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of host genomic DNA (hgDNA) contamination. Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA but are cell membrane-impermeable and thus are unable to bind protected DNA and RNA such as viral genomic material. DNA modified by EMA or PMA is not amplifiable by polymerase. In order to assess the capacity of EMA or PMA to lower hgDNA, serum or lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with different combination of treatment: with or without ultracentrifugation and incubation with or without different concentration of EMA or PMA. PVDA and HTS were used to evaluate the capacity of both techniques to detect the presence of PRRSV from each sample. Negative results were obtained by PVDA and low amount of PRRSV specific reads were obtained by HTS with untreated samples or samples treated only by ultracentrifugation. An increase capacity of PRRSV detection was observable by PVDA in EMA and PMA treated samples but PVDA best results were obtained following PMA treatment, with or without ultracentrifugation. HTS sensitivity was also improved by a treatment with EMA or PMA, but the number of reads was significantly higher in PMA treated samples. These results support the use of PMA as a treatment to increase sensitivity of PVDA and HTS.
Project description:Investigation of whole genome gene expression level changes in porcine alveolar macrophage response to co-infection of PRRSV and M. hyopneumoniae The results in this study will be further described in Bin li et al., 2013. BMC genomics.
Project description:To investigate the gene expression level changes in the whole genome scale in porcine alveolar macrophage collected from the piglets individually or dually infected with PRRSV and Mph The results in this study will be further described in Bin li et al., 2021. Veterinary microbiology
Project description:Cell lines derived from Chlorocebus sabaeus kidney were infected with an isolate of PRRSV-1 or PRRSV-2 and ribosome profiling was performed (this entry) in parallel with RNASeq (see related accession number). These datasets were used to analyse the viral and host translatome, frameshifting on the viral genome, and putative frameshift-related ribosome pausing events. For the PRRSV-1 experiments, MA-104 cells were infected with an isolate based on the Porcilis vaccine strain (KJ127878.1 but with several accumulated mutations, see associated publication) and harvested at 8 hpi after pre-treatment with cycloheximide (CHX). For the PRRSV-2 experiments, MARC-145 cells were infected with SD95-21 PRRSV (KC469618.1), and a mutant thereof (KO2). One group of samples was harvested at 9 hpi after pre-treatment with CHX, and another group of samples was harvested at 3, 6, 9 and 12 hpi by flash-freezing without CHX pre-treatment. For all samples, RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-34 nt (PRRSV-1-infected samples, CHX-pre-treated PRRSV-2- or mock-infected samples, and non-CHX-pre-treated PRRSV-2- or mock-infected 9 hpi replicate one samples) or 19-34 nt long (all other samples). Fragments were cloned into adapters based on the TruSeq small RNA adapters. For all PRRSV-2-infected (or mock-infected) libraries, adapters with an additional seven random nucleotides at the 5′-end of the 3′-adapter and the 3′-end of the 5′-adapter were used. For PRRSV-1 replicate one, no random nucleotides were present on the adapters, and for PRRSV-1 replicate two, 14 random nucleotides were present at the 5′-end of the 3′-adapter. Libraries were sequenced on the Illumina NextSeq 500 platform as a single-end run. Non-CHX-pre-treated PRRSV-2-infected 9 hpi replicate two libraries were uploaded under a separate accession number due to differences in the size selection and sequencing protocol - see associated paired-end entry. Note that sample nomeclature (including replicate numbers) is consistent between this and the two related accessions, and RiboSeq libraries are matched with RNASeq libraries, which were prepared from the same lysate. The noCHX_Ribo_9hpi_mock_3 library is deliberately absent as this was a poor quality library.
Project description:This study describes the DNA methylation profiling using whole-genome bisulfite sequencing of mouse ES cells, either derived and maintained in 2i serum-free NDiff medium, or in the presence of serum and LIF, or maintained and derived in the presence of serum and LIF and subsequently adapted to 2i serum-free NDiff medium, or maintained and derived in the presence of 2i and LIF and subsequently adapted to 2i serum. DNA methylation profiling using whole-genome bisulfite sequencing of 14 samples, 3 different lines (E14, XT67E1, Rex/GFP-2i) of pluripotent mouse ES cells as well during conversion from 2i to serum and vice versa.
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 .