Innate response of rainbow trout gill epithelial (RTgill-W1) cell line to ultraviolet-inactivated VHSV and FliC and rhabdovirus infection

Abstract

Gills reportedly play a crucial role in induction of an antiviral immune response in fish. We investigated the expression of innate response genes in the rainbow trout gill epithelial cell line RTgill-W1 36 h after pretreatment with ultraviolet-inactivated viral hemorrhagic septicemia virus (UV-VHSV), flagellin C protein from Edwardsiella tarda (FliC), VHSV and SVCV using an Agilent 4 × 44k cGRASP salmonid microarray. RTgill-W1 cells pretreated with UV-VHSV, triggered an independent gene expression profile from those treated with a recombinant flagellin C protein from Edwardsiella tarda. In addition, exposure of RTgill-W1 cells to live viruses spring viremia of carp virus and viral hemorrhagic septicemia virus induced a less robust transcriptional change of 24 and 22 gene probes, respectively, when compared to 123 genes for UV-VHSV. Further the pretreatment of RTgill-W1 cells with (UV-VHSV) significantly reduced VHSV genome copy number at 6 d post infection (dpi) relative to the FliC-treated and untreated control. A quantitative PCR was used to study the transcriptional modulation of a set of 25 innate immune-related genes highlighted by the microarray data and a panel of 7 established antiviral genes in the protected cells. Notably, the expression of ifn1, ifn2, mx1 and mx3 were expressed more in untreated cells than in UV-VHSV-treated cells where virus replication was inhibited. The results from this study shed light on the mechanisms and pathways used by teleost gill epithelium innate immunity in combating viral and bacterial infection.

Keywords: Edwardsiella tarda; FliC; Gill epithelium; Innate immunity; Microarray; SVCV; VHSV.

Fig. 1

Scatter plot showing fitted line and confidence intervals of the correlation between expression (log2 fold) of 10 genes identified by microarray and validated with qPCR. The scatter plot demonstrates the correlation between gene expression results obtained by both methods. The estimated Pearson correlation coefficient from the plotted data was 0.9063.

Fig. 2

The differential gene expression by microarray (red) and qPCR (blue) for 10 genes examined after four treatments. The gene expression in treated cells was plotted as log2 fold change from untreated RTgill-W1 cells (negative control).

Fig. 3

Interactions between differentially expressed genes after pretreatment of RTgill-W1 cells with UV-VHSV to VHSV, SVCV, NusA, and FliC at 36 h post-treatment.

Fig. 4

The difference in VHSV load determined by quantitative PCR between cells pretreated 36 h previously with FliC, NusA or UV-VHSV at 1, 3 and 6 d following infection with VHSV. The difference in VHSV load is presented as the mean log2fold change from the untreated then infected control. A statistically significant (p < 0.5) reduction in viral load is indicated by different letters.

Fig. 5

The differential gene expression by qPCR in 25 selected genes between UV-VHSV-pretreated and untreated RTgill-W1 cells following infection with VHSV at 1, 3 and 6 dpi. Non-infected (NI) cells were included as a negative control for the expression. Genes that were significantly regulated are marked with a red triangle or blue circle to demonstrate significant higher expression in un-treated or UV-VHSV-treated cells, respectively.

Fig. 6

The differential gene expression by qPCR in selected antiviral innate markers between UV-VHSV-pretreated and untreated RTgill-W1 cells following infection with VHSV at 1, 3 and 6 dpi. Non-infected cells were included as a negative control for the expression. Genes that were significantly regulated are marked with a red triangle or blue circle to demonstrate significant higher expression in un-treated or UV-VHSV-treated cells, respectively.