Transcriptome Profiling of Oncorhynchus mykiss Infected with Low or Highly Pathogenic Viral Hemorrhagic Septicemia Virus (VHSV)

Abstract

The rainbow trout (Oncorhynchus mykiss) is the most important produced species in freshwater within the European Union, usually reared in intensive farming systems. This species is highly susceptible to viral hemorrhagic septicemia (VHS), a severe systemic disease widespread globally throughout the world. Viral hemorrhagic septicemia virus (VHSV) is the etiological agent and, recently, three classes of VHSV virulence (high, moderate, and low) have been proposed based on the mortality rates, which are strictly dependent on the viral strain. The molecular mechanisms that regulate VHSV virulence and the stimulated gene responses in the host during infection are not completely unveiled. While some preliminary transcriptomic studies have been reported in other fish species, to date there are no publications on rainbow trout. Herein, we report the first time-course RNA sequencing analysis on rainbow trout juveniles experimentally infected with high and low VHSV pathogenic Italian strains. Transcriptome analysis was performed on head kidney samples collected at different time points (1, 2, and 5 days post infection). A large set of notable genes were found to be differentially expressed (DEGs) in all the challenged groups (e.s. trim63a, acod1, cox-2, skia, hipk1, cx35.4, ins, mtnr1a, tlr3, tlr7, mda5, lgp2). Moreover, the number of DEGs progressively increased especially during time with a greater amount found in the group infected with the high VHSV virulent strain. The gene ontology (GO) enrichment analysis highlighted that functions related to inflammation were modulated in rainbow trout during the first days of VHSV infection, regardless of the pathogenicity of the strain. While some functions showed slight differences in enrichments between the two infected groups, others appeared more exclusively modulated in the group challenged with the highly pathogenic strain.

Keywords: NGS; RNA-Seq; VHS; pathogenicity; rainbow trout; transcriptomic.

Figure 1

Kaplan–Meier survival curves and qRT-PCR results. (a) Mortality challenge: survival curves of VHSV-L e VHSV-H in rainbow trout. Trial carried out by bath immersion. The y-axis reports the survival rate; the x-axis reports the observation period expressed as days post infection (dpi). Step curves represent the survival rate of challenged fish in each experimental group. (b) Transcriptomic challenge: qRT-PCR results. In the x-axis the results are grouped according to the sampling day post-infection (dpi). The y-axis shows viral target gene copy numbers (CN) detected in SPL or HK and reported on logarithmic scale. Values are expressed as means of normalized CN in 1 ng of total RNA ± SEM (n = 5). * = p-value < 0.001.

Figure 2

Principal component analysis of the normalized gene counts of the host genes. The x- and y-axes show the two dimensions that explain the overall amount of variance related to gene expression levels. Each replicate is represented by a colored numerical code based on the challenge group and the time elapsed since infection.

Figure 3

Differentially expressed gene numbers in head kidney of rainbow trout infected with VHSV High or Low virulent strains. At each time point, the total number of genes is shown in a stacked bar chart. Red and blue colors represent up- and down-regulated genes, respectively.

Figure 4

Number of common and specific genes observed after infection. Venn diagrams show: (a) the common and specific differentially expressed genes (DEGs) between different time points of VHSV-L challenged group; (b) VHSV-H challenged group; (c) between the important time points of both challenged groups. Percentages of common and specific genes reported in (a,b) are computed relative to the total number of DEGs in each time point, while in (c) they refer to the total number of DEGs overall the two infections at both time points considered.

Figure 5

GO enrichment for the L and H VHSV challenges. Dotplot representing the most specific enriched GO terms for all the analyzed time points in both infections. L1, L2, L5 refer to time points 1, 2, 5 dpi in the VHSV-L challenge; similarly, H1, H2, H5 refer to time points 1, 2, 5 dpi in the VHSV-H challenge. Statistically significant enrichments (FDR < 0.05) are presented, and the -Log FDR is shown.