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.

Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture

“…Total nucleic acid was extracted from the biomass 2 h post-induction (at 4 h of bacterial growth) and every 2 h thereafter as previously described [5,8,9,17] using the one-step protocol of Posiri et al [13] with modifications. The bacterial pellet was resuspended in 5 ml of 70% v/v ethanol in PBS, incubated at 4 °C for 5 min and collected by centrifugation at 10,000 g for 10 min at 4 °C.…”

“…The supernatant was collected, and the genetic material was allowed to precipitate at −20 °C overnight in absolute ethanol. Afterwards, the supernatant was centrifuged for 30 min at 10,000 g at 4 °C, and the formation of a white pellet was observed [5,13]. The purified dsRNA was quantified as described by García et al [5].…”

“…This mechanism silences the expression of specific genes based on homologous sequences [2]. Several new treatments based on RNAi are currently being developed, including treatments directed at controlling viral diseases in intensive animal farming systems such as aquaculture [[3], [4], [5], [6]]. Notably, several of these high-scale treatments use bacteria for dsRNA production.…”

Transgenic microalgae expressing double-stranded RNA as potential feed supplements for controlling white spot syndrome in shrimp aquaculture

“…This presented a useful opportunity to validate the dsRNA-expressing cassette, the pSS116_VP28, prior to algal transformation, and also to produce a stock of VP28 dsRNA to be used as a positive control. Building on several reports where convergent bacteriophage T7 promoters have been used to produce dsRNA in E. coli (García et al 2015; Kim et al 2015), we transformed the pSS116_VP28 vector into the RNase III deficient E. coli strain HT115(DE3). The resulting system was shown to be able to produce reasonable amount of dsRNA, but the amount was lower than that produced from hairpin expressing cassettes (Chen et al 2018).…”

Progress and limitations of dsRNA strategies in the control of viral diseases in aquaculture

“…Este ensayo preliminar estableció las bases para el uso de RNAi para el control de CyHV-3 (Gotesman et al, 2013). Recientemente, nuestro grupo ha probado con éxito la efectividad de esta estrategia contra ISAv (García et al, 2015).…”