Transcriptome analysis of epithelioma papulosum cyprini cells after SVCV infection

Abstract

Background: Spring viraemia of carp virus (SVCV) has been identified as the causative agent of spring viraemia of carp (SVC) and it has caused significant losses in the cultured common carp (Cyprinus carpio) industry. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood. In this study, deep RNA sequencing was used to analyse the transcriptome and gene expression profile of EPC cells at progressive times after SVCV infection. This study addressed the complexity of virus-cell interactions and added knowledge that may help to understand SVCV.

Results: A total of 33,849,764 clean data from 36,000,000 sequence reads, with a mean read length 100 bp, were obtained. These raw data were assembled into 88,772 contigs. Of these contigs, 19,642 and 25,966 had significant hits to the NR and Uniprot databases where they matched 17,642 and 13,351 unique protein accessions, respectively. At 24 h post SVCV infection (1.0 MOI), a total of 623 genes were differentially expressed in EPC cells compared to non-infected cells, including 288 up-regulated genes and 335 down-regulated genes. These regulated genes were primarily involved in pathways of apoptosis, oxidative stress and the interferon system, all of which may be involved in viral pathogenesis. In addition, 8 differentially expressed genes (DEGs) were validated by quantitative PCR.

Conclusions: Our findings demonstrate previously unrecognised changes in gene transcription that are associated with SVCV infection in vitro, and many potential cascades identified in the study clearly warrant further experimental investigation. Our data provide new clues to the mechanism of viral susceptibility in EPC cells.

Figure 1

Assembled contig length distribution of the Pimephales promelas transcriptome. The x-axis indicates contig size and the y-axis indicates the number of contigs of each size.

Figure 2

Gene ontology assignments for P. promelas. The annotated contigs from P. promelas sequencing that matched the three major categories, including biological process, cellular component, and molecular function were shown. The x-axis indicates the GO terms and the y-axis indicates the number of genes mapped to the indicated GO term.

Figure 3

KOG classification of the P. promelas transcriptome. A total of 12,896 predicted proteins have a KOG classification among the 25 categories. The x-axis indicates the number of predicted proteins and y-axis indicates the KOG categories.

Figure 4

KEGG classification of the P. promelas transcriptome. The x-axis indicates the number of predicted proteins and the y-axis indicates the pathway.

Figure 5

Conservation of P. promelas gene identities with other species. Number of P. promelas homologous genes identified from other species using BLASTX searches with an E- value cut-off of 10⁻⁵.

Figure 6

Gene ontology assignments for differentially expressed genes (DEGs) upon SVCV infection. The DEGs upon SVCV infection that matched various ontology (GO) categories, including biological process, cellular component, and molecular function. The x-axis indicates the GO terms and the y-axis indicates the number of genes. A, GO analysis for the up-regulated genes upon SVCV infection. B, GO analysis for the down-regulated genes upon SVCV infection.

Figure 7

KEGG classification of the differentially expressed genes (DEGs) upon SVCV infection. The KEGG classification of up-regulated genes (A) and down-regulated genes (B) upon SVCV infection is shown. The x-axis indicates the pathway and the y-axis indicates the number of DEGs.

Figure 8

RT-qPCR confirmation of the differentially expressed genes (DEGs) upon SVCV infection. Relative transcript levels (fold changes) of selected DEGs were determined by the real-time PCR, using TATA box binding protein (TBP) as the reference control, and shown by the black bars. Data shown are the mean of triplicates ± SD. Three parallel experiments were performed and one representative experiment of three is shown. The transcript abundance from DEG data is shown by the grey bars. The minus value means the gene is down-regulated after SVCV infection; while the positive value means the gene is up-regulated in SVCV-infected cells.