Transcriptome Analysis Reveals the Neuro-Immune Interactions in Duck Tembusu Virus-Infected Brain

Abstract

The duck Tembusu virus (DTMUV) is a mosquito-borne flavivirus. It causes severe symptoms of egg-drop, as well as neurological symptoms and brain damage in ducks. However, the specific molecular mechanisms of DTMUV-induced neurovirulence and host responses in the brain remain obscure. To better understand the host-pathogen and neuro-immune interactions of DTMUV infection, we conducted high-throughput RNA-sequencing to reveal the transcriptome profiles of DTMUV-infected duck brain. Totals of 117, 212, and 150 differentially expressed genes (DEGs) were identified at 12, 24, and 48 h post infection (hpi). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses uncovered genes and pathways related to the nervous system and immune responses in duck brain. Neuro-related genes, including WNT3A, GATA3, and CHRNA6, were found to be significantly downregulated. RIG-I-like receptors (DHX58, IFIH1) and Toll-like receptors (TLR2 and TLR3) were activated, inducing the expression of 22 interferon stimulated genes (ISGs) and antigen-processing and -presenting genes (TAP1 and TAP2) in the brain. Our research provides comprehensive information for the molecular mechanisms of neuro-immune and host-pathogen interactions of DTMUV.

Keywords: DTMUV; Transcriptome analysis; brain; host–pathogen interaction; molecular mechanism; neuro-immune interaction.

Figure 1

Duck Tembusu virus (DTMUV) RNA copies in the brain, liver, spleen, and kidney. DTMUV RNA copies were detected by absolute quantitative real-time PCR. Y-axis represents RNA copies in one microgram total RNA. * means p ≤ 0.05.

Figure 2

Differentially expressed genes (DEGs) identified at 12, 24, and 48 hpi. (A) Numbers of upregulated and downregulated genes at 12, 24, and 48 hpi. (B) Venn diagram of DEGs at 12, 24, 48 hpi. The image shows the numbers of common and unique DEGs.

Figure 3

Twenty-four common genes shared by 12, 24, and 48 hpi. The number represents log₂ (fold change) of each gene at different time points.

Figure 4

Gene ontology (GO) enrichment analysis of DEGs (p < 0.05). (A) Ten GO terms significantly enriched at 12 hpi. (B) Thirty GO terms significantly enriched at 24 hpi. (C) Twelve GO terms significantly enriched at 48 hpi. The red portion of the bar represents the number of upregulated genes, and the blue portion of the bar represents the number of downregulated genes.

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs (p < 0.05). (A) Six KEGG terms were significantly enriched at 12 hpi. (B) Forty KEGG terms were significantly enriched at 24 hpi. (C) Twenty-four KEGG terms were significantly enriched at 48 hpi. The red portion of the bar represents the number of upregulated genes, and the blue portion of the bar represents the number of downregulated genes.

Figure 6

GO terms related to the nervous system (p < 0.05). (A) Negative regulation of neuron apoptotic process was significantly enriched at 12 hpi. (B) Axon guidance, postsynaptic membrane, and oligodendrocyte differentiation were significantly enriched at 24 hpi. (C) Synapse was significantly enriched at 48 hpi. The X-axis represents log₂ (fold change), and the Y-axis represents genes enriched in each GO term. The red bar represents the upregulated gene, and the green bar represents the downregulated gene.

Figure 7

KEGG terms related to nervous system (p < 0.05). (A) Neuroactive ligand–receptor interaction and the axon guidance pathway were significantly enriched at 24 hpi. (B) Retrograde endocannabinoid signaling and the GABAergic synapse pathway were significantly enriched at 48 hpi. The X-axis represents log₂ (fold change), and the Y-axis represents genes enriched in each KEGG pathway. The red bar represents the upregulated gene, and the green bar represents the downregulated gene.

Figure 8

Validation of RNA-seq by qRT-PCR. The X-axis represents hours post infection (hpi). The Y-axis represents fold change. * means p ≤ 0.05, ** means p ≤ 0.01, *** means p ≤ 0.001.