Transcriptome Analysis of Duck and Chicken Brains Infected with Aquatic Bird Bornavirus-1 (ABBV-1)

Abstract

Aquatic bird bornavirus 1 (ABBV-1) is a neurotropic virus that infects waterfowls, resulting in persistent infection. Experimental infection showed that both Muscovy ducks and chickens support persistent ABBV-1 infection in the central nervous system (CNS), up to 12 weeks post-infection (wpi), without the development of clinical disease. The aim of the present study was to describe the transcriptomic profiles in the brains of experimentally infected Muscovy ducks and chickens infected with ABBV-1 at 4 and 12 wpi. Transcribed RNA was sequenced by next-generation sequencing and analyzed by principal component analysis (PCA) and differential gene expression. The functional annotation of differentially expressed genes was evaluated by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The PCA showed that the infected ducks sampled at both 4 and 12 wpi clustered separately from the controls, while only the samples from the chickens at 12 wpi, but not at 4 wpi, formed a separate cluster. In the ducks, more genes were differentially expressed at 4 wpi than 12 wpi, and the majority of the highly differentially expressed genes (DEG) were upregulated. On the other hand, the infected chickens had fewer DEGs at 4 wpi than at 12 wpi, and the majority of those with high numbers of DEGs were downregulated at 4 wpi and upregulated at 12 wpi. The functional annotation showed that the most enriched GO terms were immune-associated in both species; however, the terms associated with the innate immune response were predominantly enriched in the ducks, whereas the chickens had enrichment of both the innate and adaptive immune response. Immune-associated pathways were also enriched according to the KEGG pathway analysis in both species. Overall, the transcriptomic analysis of the duck and chicken brains showed that the main biological responses to ABBV-1 infection were immune-associated and corresponded with the levels of inflammation in the CNS.

Keywords: GO; KEGG; aquatic bird birnavirus-1; bornaviruses; lncRNA; transcriptomics.

Figure 1

Principal component analysis (PCA) plot of Muscovy duck (A) and chicken (B) cohorts infected with ABBV−1. Light brown and dark brown represent control birds from 4 and 12 wpi, respectively. Light blue and dark bluerepresent infected birds from 4 and 12 wpi, respectively. The alphanumeric code adjacent to each data point represents a single bird identifier. For each species, there were 7 birds per treatment per time point. Red circle shows clustering of infected animals and blue circle shows clustering of control animals. Green circle shows mixture of infected and control animals.

Figure 2

Overview of viral RNA copies and pathology scores in the brains of ducks and chickens alongside total and highly differentially expressed genes in those tissues. In (A), data are presented for Muscovy ducks at 4 wpi and 12 wpi. In (B), data are presented for chickens at 4 wpi and 12 wpi. The left y-axis shows both the log₁₀ RNA copies and pathology scores. The right y-axis shows the number of differentially expressed genes. The x-axis shows the weeks post-infection. tDEG stands for total differentially expressed genes and hDEG stands for highly differentially expressed genes. The viral-RNA and pathology-score data used in this figure are adapted from [15,16].

Figure 3

Venn diagram showing proportions of highly differentially expressed genes (hDEGs) between 4 and 12 weeks post-infection (wpi), in both Muscovy ducks and chickens. In (A), hDEGs in the brains of ABBV-1-infected Muscovy ducks at 4 wpi were compared with those at 12 wpi. The majority of hDEGs occurred at 4 wpi, and 217 hDEGs were shared between 4 and 12 wpi. In (B), the hDEGs in the brains of ABBV-1-infected chickens at 4 wpi were compared with those at 12 wpi. The majority of hDEGs occurred at 12 wpi, and only 11 hDEGs were shared between the two time points.

Figure 4

Gene ontology (GO) analysis of Muscovy duck and chicken brains infected with ABBV−1 at 4 and 12 weeks post-infection (wpi). In (A), the 20 highest-ranked (out of 59) and 14 (out of 14) enriched GO terms are shown for the Muscovy ducks sampled at 4 wpi (left graph) and 12 wpi (right graph), respectively. In (B), the 20 highest-ranked (out of 107) significantly enriched GO terms are shown for the chickens sampled at 12 wpi. Chicken brains sampled at 4 wpi did not produce any significantly enriched GO terms. The significance of the GO terms’ enrichment is represented as −log₂ of the adjusted p-values on the x-axis. The GO categories are labeled and grouped by colors (y-axis): red bars represent biological processes, green bars represent cellular components, and blue bars represent molecular function. The numbers inside the bars represent the numbers of differentially expressed genes per GO term.