Project description:Background: Infection with Turkey Hemorrhagic Enteritis Virus (THEV) can cause hemorrhagic enteritis, which affects turkey poults. This disease is characterized by immunosuppression (IMS) and bloody droppings. The clinical disease usually lasts only a few days but secondary opportunistic infections due to THEV-induced IMS extend the duration of illness and mortality, which exacerbates economic losses. Although an avirulent THEV strain with only subclinical disease is used as a vaccine, some immunosuppressive properties remain in this prophylactic strain. To elucidate the mechanisms mediating THEV-induced IMS, we performed the first transcriptomic analysis of a THEV infection using bulk RNA-sequencing. Methods: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection (hpi). Total RNA was extracted, and poly-Adenylated-tailed mRNAs were sequenced. Reads were mapped to the host turkey genome after trimming and gene expression was quantified with StringTie. Differential gene expression was performed with DESeq2 followed by functional enrichment analysis with gprofiler2 and DAVID from NCBI. RT-qPCR of select genes was performed to validate the RNA-sequencing data. Results: A total of 2,343 and 3,295 differentially expressed genes (DEGs) were identified at 12 hpi and 24 hpi, respectively. At 12 hpi, 1,079 genes were upregulated and 1,264 genes downregulated, whereas 1,512 genes were upregulated and 1,783 genes downregulated at 24 hpi. The DEGs contributed to multiple biological processes including apoptosis, ER unfolded protein response, and cell maintenance. Multiple pro-apoptotic genes, including APAF1, BNIP3L, BMF, BAK1, RIPK1, and FAS were upregulated. Genes that play a role in ER stress-induced unfolded protein response including VCP, UFD1, EDEM1, EDEM3, and ATF4 were also upregulated and may contribute to apoptosis. Conclusions: Our data suggest that several biological processes and pathways including apoptosis, immune response, ER response to stress, ubiquitin-dependent protein catabolic process, and repression of essential cellular maintenance are important aspects of the host cell response to THEV infection. It is possible that interplay between multiple processes may mediate apoptosis of infected B-cells, leading to IMS.

Project description:BackgroundHemorrhagic enteritis, caused by Turkey Hemorrhagic Enteritis Virus (THEV), is a disease affecting turkey poults characterized by immunosuppression and bloody diarrhea. An avirulent THEV strain that retains the immunosuppressive ability is used as a live vaccine. Characterizing the splice map of THEV is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used RNA sequencing to characterize the splice map of THEV for the first time, providing key insights into the THEV gene expression and mRNA structures.MethodsAfter infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions.ResultsResearchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3' rapid amplification of cDNA ends (3' RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated.ConclusionsSimilar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs.

Project description:The success of turkey breeding for rapid growth rate and larger breast size has coincided with an increasing incidence of a meat quality defect described as pale, soft and exudative (PSE). We hypothesized that this defect, which is associated with an abnormally rapid rate of postmortem metabolism, derives from altered expression of genes involved in metabolic regulation. Our objective was to use deep transcriptome RNA sequence analysis (RNAseq) to identify differentially expressed genes between normal and PSE turkey breasts. Following harvest of turkey breasts (n = 43), the pH at 15 min post-slaughter and percent marinade uptake at 24h post-slaughter were determined. Breast samples were classified as normal or PSE based on marinade uptake (high = normal; low = PSE). Total RNA from samples with the highest (n=4) and lowest (n=4) marinade uptake were isolated and sequenced using the Illumina GAIIX platform. Of 21,340 gene loci discovered by RNAseq, 8480 loci completely matched the turkey reference genome, and 480 genes were differentially expressed (false discovery rate, FDR<0.05) between normal and PSE samples. Two highlights were the genes nephroblastoma overexpressed (NOV), upregulated about 38-fold and pyruvate dehydrogenase kinase isoform 4 (PDK4), downregulated 14-fold in PSE samples. Pathway analysis suggested that several biological functions, including carbohydrate metabolism and energy production, were affected by meat quality. Because PDK4 regulates conversion of pyruvate to acetyl CoA, differences in regulation of oxidative metabolism may exist among turkeys. Accelerated early postmortem metabolism would result in faster pH decline in PSE meat. This hypothesis was supported by the fact that decreased expression of PDK4 was associated with lower pH in PSE samples (pH[PSE] = 5.59M-BM-10.09, pH[normal] = 5.77M-BM-10.17). The RNAseq results provided a greater molecular mechanistic understanding of development of PSE turkey, which will be a foundation for new intervention strategies to prevent development of this defect. The mRNA profiles of normal and PSE turkey breast muscle were generated by deep sequencing using Illumina GAIIx platform. Multiplexing was performed (2 samples/lane). Afterwards, difference in gene expression between normal and PSE samples were tested.

Project description:The success of turkey breeding for rapid growth rate and larger breast size has coincided with an increasing incidence of a meat quality defect described as pale, soft and exudative (PSE). We hypothesized that this defect, which is associated with an abnormally rapid rate of postmortem metabolism, derives from altered expression of genes involved in metabolic regulation. Our objective was to use deep transcriptome RNA sequence analysis (RNAseq) to identify differentially expressed genes between normal and PSE turkey breasts. Following harvest of turkey breasts (n = 43), the pH at 15 min post-slaughter and percent marinade uptake at 24h post-slaughter were determined. Breast samples were classified as normal or PSE based on marinade uptake (high = normal; low = PSE). Total RNA from samples with the highest (n=4) and lowest (n=4) marinade uptake were isolated and sequenced using the Illumina GAIIX platform. Of 21,340 gene loci discovered by RNAseq, 8480 loci completely matched the turkey reference genome, and 480 genes were differentially expressed (false discovery rate, FDR<0.05) between normal and PSE samples. Two highlights were the genes nephroblastoma overexpressed (NOV), upregulated about 38-fold and pyruvate dehydrogenase kinase isoform 4 (PDK4), downregulated 14-fold in PSE samples. Pathway analysis suggested that several biological functions, including carbohydrate metabolism and energy production, were affected by meat quality. Because PDK4 regulates conversion of pyruvate to acetyl CoA, differences in regulation of oxidative metabolism may exist among turkeys. Accelerated early postmortem metabolism would result in faster pH decline in PSE meat. This hypothesis was supported by the fact that decreased expression of PDK4 was associated with lower pH in PSE samples (pH[PSE] = 5.59±0.09, pH[normal] = 5.77±0.17). The RNAseq results provided a greater molecular mechanistic understanding of development of PSE turkey, which will be a foundation for new intervention strategies to prevent development of this defect.

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Turkey B-cell Transcriptome Profile During Turkey Hemorrhagic Enteritis Virus (THEV) Infection Highlights Upregulated Apoptosis and Breakdown Pathways That May Mediate Immunosuppression

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