Temporal changes in transcriptome profile provide insights of White Spot Syndrome Virus infection in Litopenaeus vannamei

Abstract

Shrimp aquaculture is severely affected by WSSV. Despite an increasing effort to understand host/virus interaction by characterizing changes in gene expression (GE) following WSSV infection, the majority of published studies have focussed on a single time-point, providing limited insight on the development of host-pathogen interaction over the infection cycle. Using RNA-seq, we contrasted GE in gills of Litopenaeus vannamei at 1.5, 18 and 56 hours-post-infection (hpi), between WSSV-challenged and control shrimps. Time course analysis revealed 5097 differentially expressed genes: 63 DEGs were viral genes and their expression in WSSV group either peaked at 18 hpi (and decreased at 56 hpi) or increased linearly up to 56 hpi, suggesting a different role played by these genes during the course of infection. The remaining DEGs showed that WSSV altered the expression of metabolic, immune, apoptotic and cytoskeletal genes and was able to inhibit NF-κB and JAK/STAT pathways. Interestingly, GE changes were not consistent through the course of infection but were dynamic with time, suggesting the complexity of host-pathogen interaction. These data offer novel insights into the cellular functions that are affected during the course of infection and ultimately provide a valuable resource towards our understanding of the host-pathogen dynamics and its variation with time.

Figure 1

Viral load. The number of copies of WSSV detected at different time points after injection of WSSV in L. vannamei (dots represent the average copy number from n = 3 independent biological replicates per time point).

Figure 2

Time Course Analysis. Graphs represent the 9 clusters of expression in which the 5097 differentially expressed genes (in L. vannamei infected or non-infected) were divided based on their expression profile. In each graph, dots represent the median expression level for each biological replicate in each time point (n = 3 different biological replicates for each treatment (e.g. Ctrl and WSSV infected) for each time point (e.g. 1.5 h, 18 h and 56 h)). For each treatment, the average expression levels in each time point are connected by a line. Green dots and lines = Ctrl while purple dots and lines = WSSV infected.

Figure 3

Differentially expressed viral genes from TC analysis. (A) Boxplots of differentially expressed viral genes (in L. vannamei infected or non-infected) with a bell-shaped profile of expression with time (e.g. between 1.5 h and 56 h). Each symbol represents the average expression level (as trimmed means of M values (TMM)) of a single viral gene. (B) Boxplots of differentially expressed viral genes with a linear expression profile with time (e.g. between 1.5 h and 56 h). Each symbol represents the average expression level (as trimmed means of M values (TMM)) of a single viral gene. A list of all DE viral contigs is found in Suppl. Table 5.

Figure 4

Heatmap of ABC transporter DE genes and cytoskeletal DE genes. Heatmap showing DE genes (in L. vannamei infected or non-infected) mapping to ABC transporter genes and cytoskeletal genes in control (“Ctrl”) and WSSV infected (“WSSV”) animals at different time points (e.g. 1.5 h, 18 h, 56 h). Each cell in the heatmap represents the average expression level from three independent biological replicate samples. Colour legend is on a log10 scale. Trinity contig names matching each gene can be found in Suppl. Table 6.

Figure 5

Heatmap of DE genes associated with metabolism. Heatmap showing DE genes (in L. vannamei infected or non-infected) mapping to three metabolic processes (e.g. Glycolysis, Oxidative phosphorylation and Kreb cycle) in control (“Ctrl”) and WSSV infected (“WSSV”) animals at different time points (e.g. 1.5 h, 18 h and 56 h). Each cell in the heatmap represents the average expression level from three independent biological replicate samples. Colour legend is on a log10 scale. Trinity contig names matching each gene can be found in Suppl. Table 6.

Figure 6

Heatmap of immune DE genes. Heatmap showing DE genes (in L. vannamei infected or non-infected) mapping to immune genes in control (“Ctrl”) and WSSV infected (“WSSV”) animals at different time points (e.g. 1.5 h, 18 h and 56 h). Each cell in the heatmap represents the average expression level from three independent biological replicate samples. Colour legend is on a log10 scale. Trinity contig names matching each gene can be found in Suppl. Table 6.

Figure 7

Heatmap of DE genes associated with immune pathways. Heatmap showing DE genes (in L. vannamei infected or non-infected) mapping to immune pathways (e.g. NF-kB pathway and JAK/STAT pathway) in control (“Ctrl”) and WSSV infected (“WSSV”) animals at different time points (e.g. 1.5 h, 18 h and 56 h). Each cell in the heatmap represents the average expression level from three independent biological replicate samples. Colour legend is on a log10 scale. Trinity contig names matching each gene can be found in Suppl. Table 6.

Figure 8

Heatmap of DE genes associated with apoptosis. Heatmap showing DE genes (in L. vannamei infected or non-infected) mapping to apoptotic processes in control (“Ctrl”) and WSSV infected (“WSSV”) animals at different time points (e.g. 1.5 h, 18 h and 56 h). Each cell in the heatmap represents the average expression level from three independent biological replicate samples. Colour legend is on a log10 scale. Trinity contig names matching each gene can be found in Suppl. Table 6.

Figure 9

Confirmatory qPCRs of selected DE genes. Normalised Relative Quantities of selected genes from the DE genes using Elongation factor as housekeeping gene at each time point (e.g. 1.5 h, 18 h and 56 h). Data are shown as mean + SD of n = 3 independent biological replicates. Green columns represent Ctrl values, while purple columns represent WSSV infected values.