Inactivated E. coli transformed with plasmids that produce dsRNA against infectious salmon anemia virus hemagglutinin show antiviral activity when added to infected ASK cells

Abstract

Infectious salmon anemia virus (ISAV) has caused great losses to the Chilean salmon industry, and the success of prevention and treatment strategies is uncertain. The use of RNA interference (RNAi) is a promising approach because during the replication cycle, the ISAV genome must be transcribed to mRNA in the cytoplasm. We explored the capacity of E. coli transformed with plasmids that produce double-stranded RNA (dsRNA) to induce antiviral activity when added to infected ASK cells. We transformed the non-pathogenic Escherichia coli HT115 (DE3) with plasmids that expressed highly conserved regions of the ISAV genes encoding the nucleoprotein (NP), fusion (F), hemagglutinin (HE), and matrix (M) proteins as dsRNA, which is the precursor of the RNAi mechanism. The inactivated transformed bacteria carrying dsRNA were tested for their capacity to silence the target ISAV genes, and the dsRNA that were able to inhibit gene expression were subsequently tested for their ability to attenuate the cytopathic effect (CPE) and reduce the viral load. Of the four target genes tested, inactivated E. coli transformed with plasmids producing dsRNA targeting HE showed antiviral activity when added to infected ASK cells.

Keywords: antiviral; aquaculture; bacterial delivery; double-stranded RNA; infectious salmon anemia virus.

Figure 1

Co-localization assay of E. coli HT115 expressing GFP in ASK cells. Green E. coli were added to ASK cells stained with fluorescent orange dye. Phase contrast (upper panel) and fluorescence (lower panel) confocal microscopy images were taken at 24 h (left) and 48 h (right). Bacteria were visualized as orange/yellow when they co-localized with the orange fluorescent probe in the cell cytoplasm, as indicated with arrows at 48 h in the right bottom panel and an asterisk in the enlarged panel. Scale bars represent 10 μm.

Figure 2

Relative expression of viral mRNA in infected ASK cells treated with different dsRNAs. (A) Relative viral mRNA expression of NP, F, HE, and M in ASK cells infected with ISAV HPR35 and treated with HT115 E. coli carrying dsRNA against NP (dsRNA NP), F (dsRNA F), HE (dsRNA HE), or M (dsRNA M). Data were compared with viral mRNA expression of infected but non-treated ASK cells and expressed as fold change in log₂. (B) Relative expression of HE mRNA in ASK cells infected with ISAV HPR35 and treated with HT115 E. coli carrying dsRNA against HE (dsRNA HE), a mixture of the four E. coli strains carrying each of the dsRNAs (dsRNA NP/F/HE/M) or E. coli carrying the empty vector (pL4440) compared with viral mRNA expression in infected but non-treated ASK cells. Data are expressed as fold change in log₂. Bars represent the average results (n = 3), and the error bars represent standard errors of the means (SEM). (^***) indicates significant differences between treated, infected ASK cells vs. untreated, infected ASK cells, p < 0.05. ^**indicates significant differences between NP/F/HE/M-mix treated, infected ASK cells vs. HE treated, infected ASK cells, p < 0.05.

Figure 3

Expression of HE protein in infected ASK-cells treated with dsRNA. Lysates from ASK cells infected with HPR35 ISAV, infected ASK cells treated with E. coli HT115 carrying dsRNA against HE (dsRNA HE), the dsRNA NP/F/HE/M mixture or empty vector (pL4440) were analyzed by western blot using HE-antibody for viral protein detection. All data were normalized to β-actin expression (HE/β-Actin). Bars represent the average result (n = 3), and the error bars represent standard errors of the means (SEM). (^**) indicates a significant difference between infected ASK cells treated with dsRNA against HE (dsRNA HE) vs. ASK infected cells (ISAV HPR35), and infected ASK cells treated with the empty vector (pL4440), p < 0.05.

Figure 4

Effect of HE dsRNA on viral titer (PFU/mL) in the supernatants of infected ASK cells. Viral titers (PFU/mL) of ISAV in the supernatants of ASK cells infected with ISAV HPR35 (HPR35), infected ASK cells treated with E. coli HT115 carrying dsRNA against HE (dsRNA HE) or empty vector (pL4440). The supernatant of uninfected cells was added as an assay control (ASK). Bars represent the average result (n = 3), and the error bars represent standard errors of the means (SEM). (^*) indicates a significant difference between HPR35-infected ASK cells treated with dsRNA against HE (dsRNA HE) vs. infected ASK cells (HPR35), p < 0.05.

Figure 5

The effect of HE dsRNA on induced CPEs in infected ASK cells. (A) Uninfected ASK cells at 11 days post-infection (B) ASK cells infected with HPR35 ISAV without treatment and (C) with the addition of E. coli carrying dsRNA against HE. (D) Infected ASK cells and treatment with E. coli HT1115 carrying empty vector pL4440 was included as a control. The CPEs observed in ASK cells infected as described were vacuolated cells and monolayer detachment. The scale bar represents 20 μm.