Influence of Viral Re-Infection on Head Kidney Transcriptome of Nervous Necrosis Virus-Resistant and -Susceptible European Sea Bass (Dicentrarchus labrax, L.)

Abstract

Fish viral infections have great environmental and economic implications in aquaculture. Nervous necrosis virus (NNV) is a pathogen affecting more than 120 different species, causing high mortality and morbidity. Herein, we study how NNV re-infection affects the European sea bass (Dicentrarchus labrax, L.) head kidney transcriptome in disease-resistant and -susceptible sea bass families. To determine how each family responds to re-infection, we performed the RNA-sequencing analysis of experimentally NNV-infected D. labrax. Fish were experimentally infected in a long-term study, and one month after the last recorded death, all surviving fish were re-infected by the same NNV strain. Fish tissues were sampled 7 days upon re-infection. The transcriptome profiles of infected vs. non-infected fish revealed 103 differentially expressed genes (DEGs) for the resistant family and 336 DEGs for the susceptible family. Only a few pathways were commonly enriched in the two families, further indicating that the resistant and susceptible families utilize completely different mechanisms to fight the NNV re-infection. Protein-protein interaction analysis identified a variety of hub genes for the resistant and the susceptible families, quite distinct in their function on NNV resistance. In conclusion, NNV-resistant and -sensitive sea bass transcriptomes were analyzed following NNV survivors' viral re-infection, offering a glimpse into how host attempts to control the infection depending on its genetic background in relation with virus resistance.

Keywords: European sea bass; disease resistance; host–pathogen interaction; nervous necrosis virus; nodavirus; transcriptome; viral nervous necrosis; viral re-infection.

Figure 1

Experimental setup (NNV: nervous necrosis virus; R: resistant; RI: resistant infected; RNI: resistant non-infected; S: susceptible; SI: susceptible infected; SNI: susceptible non-infected; D: days post infection).

Figure 2

Cumulative survival of NNV-challenged resistant (continuous blue line) and susceptible (dashed magenta line) sea bass families. The mean mortality is represented in each time point (days post infection).

Figure 3

(A) Statistics of differentially expressed genes (DEGs) in RI vs. RNI, SI vs. SNI, SI vs. RI, and SNI vs. RNI groups. The X axis and Y axis represent a comparison of samples and the number of DEGs. The first column at each time-point graph corresponds to up-regulated genes, and the second column corresponds to down-regulated genes. (B) Venn diagram showing the number of common and exclusive DEGs in RI vs. RNI, SI vs. SNI, SI vs. RI, and SNI vs. RNI groups. (C) Cluster analysis of DEGs. Heat map of the genes showed a more than 1.5-fold difference between the resistant (R: blue bar) and susceptible (S: magenta bar) families in NNV-infected (I: orange bar) and non-infected (NI: green bar) experimental groups. Red and blue colors indicate up- and down-regulation in Log2 cpm values, respectively. Each column in the graph represents a sample, each row represents a gene, and the expression of genes in different samples is represented by different colors, with redder colors indicating higher expression and bluer colors indicating lower expression.

Figure 4

The top 30 gene ontology (GO)-enriched terms in RI vs. RNI groups: (A) up-regulated and (B) down-regulated DEGs. BP: biological process, MF: molecular function, CC: cellular component.

Figure 5

(A) The top 30 enriched KEGG pathways in RI vs. RNI groups. (B) The enriched Reactome pathways in RI vs. RNI groups. ST: signal transduction; DB: developmental biology; PCD: programmed cell death; ImS: immune system; H: hemostasis; NES: normalized enrichment score.

Figure 6

Protein–protein interaction (PPI) networks, with k-means clustering for the zebrafish orthologs of the differentially expressed genes in D. labrax RI vs. RNI groups. It is retrieved via API access to the STRING database (https://string-db.org) (accessed on 27 November 2024) and was performed based on the Danio rerio protein database. Each colored group represents a different cluster. The edges represent protein–protein interactions.

Figure 7

The gene ontology (GO)-enriched terms in SI vs. SNI groups: (A) up-regulated and (B) down-regulated DEGs. BP: biological process, MF: molecular function, CC: cellular component.

Figure 8

(A) The top enriched KEGG pathways in SI vs. SNI groups. (B) The enriched Reactome pathways in SI vs. SNI groups. ImS: immune system, Met: metabolism, ADME: drug ADME, GE: gene expression (transcription), ST: signal transduction, NES: normalized enrichment score.

Figure 9

Protein–protein interaction (PPI) networks, with k-means clustering for the zebrafish orthologs of the differentially expressed genes in D. labrax SI vs. SNI groups. It is retrieved via API access to the STRING database (https://string-db.org) (accessed on 27 November 2024) and was performed based on the Danio rerio protein database. Each colored group represents a different cluster. The edges represent protein-protein interactions.