Project description:The host response of the primary intestinal epithelium to human astrovirus (HAstV infection has not been elucidated to date. In order to characterize the global effects of AstV infection on human intestinal tissue, we performed transcriptional profiling of VA1-infected human intestinal enteroids (HIE) by RNAseq. We used the D124 line for our studies since AstV infections are typically symptomatic in very young children, and our prior infection studies indicated rapid infection in D124. D124 HIE were mock-infected or infected with VA1 (MOI = 1) and harvested at 0 hpi (i.e., 1h post-adsorption), 12 hpi, and 24 hpi (Fig 4A). To monitor viral replication, VA1 genome copies were detected by RT-qPCR, revealing an approximately 1 and 2 log increase at 12 and 24 hpi, respectively (Fig 4B). Cellular RNA was extracted and analyzed by RNAseq. Genome copies as determined by RT-qPCR were correlated to the proportion of viral transcripts in the pool of sequenced RNA collected from the same HIE cultures, revealing an exceptionally strong correlation between the two measures of viral replication (r2 = 0.98, P = 2.5 x 10^-14. VA1 genome reads contributed 0.05 ± 0.02 x% of total RNAseq reads at 24 hpi, further confirming robust infection. Differential expression analysis was performed to identify genes associated with the HIE host response to VA1 infection. A total of 23,220 genes were detected. Differential expression of genes at each timepoint was graphed in a volcano plot. The log2 fold change in normalized expression (transcripts per million reads [TPM]) of all expressed host genes in VA1-infected HIEs relative to mock-infected HIE is shown on the x-axis. The -log10 transformed P-value is given on the y-axis. Genes that are significantly up-regulated (adjusted P < 0.05) in VA1-infected D124 HIE relative to mock-infected HIE are colored red, while significantly down-regulated genes are colored blue. Overall, after a 1 hour adsorption (0 hpi), 110 genes were significantly upregulated and 136 genes were downregulated , indicating changes due to viral attachment to cells. This number was reduced at 12 hpi, with 8 significantly upregulated and 5 downregulated genes. At 24 hpi, 154 upregulated and 49 downregulated genes compared to the mock-infected control were identified. We next identified the top 15 significantly up- and down-regulated genes at 24 hpi. This group of genes was used to generated a heatmap of the mean scaled fold-change (Z-score) in expression of each of them in virus-infected HIE relative to mock-infected HIE at each timepoint (Fig 4D). Most of the upregulated genes at 24 hpi were involved in type I and type III interferon (IFN) signaling. Of the IFN genes, IFNL1 was highly upregulated, with IFNA1 and IFNB1 upregulation being slightly lower (Fig S4B). No upregulation was observed for the genes encoding IFN-γ, or the type I and III IFN receptors (data not shown). The top 12 IFN-stimulated genes (ISGs) also positively correlated with VA1 infection (Fig S4C). Conversely, the top 12 downregulated genes, including fermitin family member 1 (FERMT1), signal peptide peptidase like 3 (SPPL3), and tetratricopeptide repeat domain 19 (TTC19) negatively correlated with VA1 infection (Fig S4D). Next, we evaluated lists of the top 100 up- and down-regulated genes at 24 hpi using an over-abundance test to identify significantly over-represented REACTOME pathways in these lists. These data revealed that the top four significantly enriched pathways among upregulated genes were all related to innate antiviral signaling (Fig 4E), which will be investigated in more detail below. For downregulated genes, the top two pathways were “neurexins and neuroligins”, which play signaling roles in synapse development, and “protein-protein interactions at synapses”. The biological significance of synapses during astrovirus infection remains to be elucidated. In order to evaluate the potential for coordinated and directional activation of genes in known signaling pathways, we applied gene set enrichment analysis (GSEA) to our RNAseq differential expression data. Based on the strong dominance of IFN signaling pathways, we focused our GSEA analysis on immune signaling (Fig 4F). During the adsorption phase, nucleic acid pattern recognition receptor signaling pathways (TLRs, STING) were upregulated, consistent with their early role in virus recognition and induction of IFN signaling. At 24 hpi, these early signaling events had been largely replaced by the later phase of IFN signaling and expression of ISGs. Taken together, these data indicate that VA1 infection predominantly elicited antiviral IFN signaling in HIE-derived fetal duodenum at the transcript level.

Project description:Rainbow trout (Oncorhynchus mykiss) is a species of cold-water fish with important economic values, which is widely cultivated around the world. It is susceptible to infectious hematopoietic necrosis virus (IHNV), resulting in a large number of deaths. In this study, we measured the changes of immune parameters in different periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 hours post infection (hpi)), transcriptome profiles of T48G/C48G and important immune-related genes expression patterns in rainbow trout gill following IHNV challenge through biochemical methods, RNA-seq and qRT-PCR. The results showed that AKP, ACP, T-SOD, ALT and AST activities, as well as MDA and LZM content decreased and then increased during infection, and reached to the lowest value at 48 hpi (P < 0.05), whereas CAT activity showed significant peaks at 48 hpi (P < 0.05). The mRNA and miRNA analysis identified 4343 differentially expressed genes (DEGs) and 11 differentially expressed miRNAs (DEMs), and numerous DEGs involved in immune responses, such as tlr3, tlr7, tlr8, traf3, ifih1, trim25, dhx58, ddh58, hsp90a.1, nlrc3, nlrc5, socs3, irf3, irf7, mx1, vig2, bcl2, casp3 and casp8。Integrated analysis identified four key miRNAs (miR-27d-3p, miR-124-5p, miR-301a-5p and miR-146-5p) that target important immune-related DEGs (tlr3, ifih1, vig2, irf8, socs3 and cox1). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs and target genes were significantly enriched in various immune-related terms including inflammatory response, DNA replication, cell adhesion and pathways of Toll-like receptor signaling, apoptosis, DNA replication, p53 signaling and MAPK signaling. The expression trends of 15 DEGs and 2 DEMs were further verified by quantitative real-time PCR, which confirmed the reliability of the transcriptome sequencing results. Expression analysis showed that tlr3, tlr7, traf3, ifih1, trim25, dhx58, hap90a.1, irf3, irf7 and mx1 genes increased significantly after 48 hpi during infection. This study contributes to the understanding of the immune response of rainbow trout against IHNV infection, and provides new insights into the immune regulation mechanisms for future studies.

Project description:Rainbow trout (Oncorhynchus mykiss) is an important economical cold-water fish worldwide. However, infection with infectious hematopoietic necrosis virus (IHNV) has severely restricted the development of aquaculture and caused huge economic losses. Currently, little is known about the immune defense mechanisms of rainbow trout against IHNV. In this study, we detected the changes of immune parameters over different post-infection periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 hours post-infection (hpi)), mRNA and miRNA expression profiles under 48 hpi (T48L) compared to control (C48L), and key immune-related genes expression patterns in rainbow trout liver following IHNV challenge through biochemical methods, RNA-seq, and qRT-PCR, and the function of miR-330-y was verified by overexpression and silencing in vitro and in vivo. The results revealed that AKP, ALT, CAT, and T-SOD activities, and LZM content showed significant peaks at 48 hpi (P < 0.05), whereas MAD content and AST activity decreased continuously during infection (P < 0.05), and ACP activity varied slightly. From RNA-seq, a total of 6844 genes and 86 miRNAs were differentially expressed, and numerous immune-related differentially expressed genes (DEGs) involved in RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and antigen processing and presentation were significantly upregulated in T48Lm group, including IFIH1, DHX58, MAVS, TRAF3, IRF3, IRF7, MX1, TLR3, TLR8, MYD88, NOD1, NOD2, IL-8, CXCR1, CD209, CD83, and TAP1. Integrated analysis identified seven miRNAs (miR-425-x, miR-185-x, miR-338-x, miR-330-y, miR-361-x, miR-505-y, and miR-191-x) that target at least three key immune-related DEGs. Expression analysis showed that IFIH1, DHX58, IRF3, IRF7, MX1, TLR3, TLR8, and MYD88 showed a marked increase after 24 hpi during infection. Further research confirmed TAP1 as one of the targets of miR-330-y, overexpression of miR-330-y with mimics or agomir significantly reduced the expression levels of TAP1, IRF3, and IFN, and the opposite effects were obtained by inhibitor. These results facilitate in-depth understanding of the immune mechanisms in rainbow trout against IHNV and will provide valuable information for disease resistance breeding.

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Time-Course Transcriptome Analysis of Lungs From Mice Infected With Hypervirulent Klebsiella pneumoniae via Aerosolize Intratracheal Inoculation

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