Transgenically mediated shRNAs targeting conserved regions of foot-and-mouth disease virus provide heritable resistance in porcine cell lines and suckling mice

Abstract

Foot-and-mouth disease virus (FMDV) is responsible for substantial economic losses in livestock breeding each year, and the development of new strategies is needed to overcome the limitations of existing vaccines and antiviral drugs. In this study, we evaluated the antiviral potential of transgenic porcine cells and suckling mice that simultaneously expressed two short-hairpin RNAs (shRNAs) targeting the conserved regions of the viral polymerase protein 3D and the non-structural protein 2B. First, two recombinant shRNA-expressing plasmids, PB-EN3D2B and PB-N3D2B, were constructed and the efficiency of the constructs for suppressing an artificial target was demonstrated in BHK-21 cells. We then integrated PB-EN3D2B into the genome of the porcine cell line IBRS-2 using the piggyBac transposon system, and stable monoclonal transgenic cell lines (MTCL) were selected. Of the 6 MTCL that were used in the antiviral assay, 3 exhibited significant resistance with suppressing ratios of more than 94% at 48 hours post-challenge (hpc) to both serotype O and serotype Asia 1 FMDV. MTCL IB-3D2B-6 displayed the strongest antiviral activity, which resulted in 100% inhibition of FMDV replication until 72 hpc. Moreover, the shRNA-expressing fragment of PB-N3D2B was integrated into the mouse genome by DNA microinjection to produce transgenic mice. When challenged with serotype O FMDV, the offspring of the transgenic mouse lines N3D2B-18 and N3D2B-81 exhibited higher survival rates of 19% to 27% relative to their non-transgenic littermates. The results suggest that these heritable shRNAs were able to suppress FMDV replication in the transgenic cell lines and suckling mice.

Biological Function and Application of Picornaviral 2B Protein: A New Target for Antiviral Drug Development

“…Furthermore, specific degradation of complementary mRNA can be triggered by small interfering RNAs (siRNAs) or folded short hairpin RNAs (shRNAs) [98], which can be explored as an RNA interference strategy, a relatively novel technology that has already been applied to treat many important pathogens, including HIV-1, hepatitis B virus, and herpes simplex virus [99,100,101]. Currently, shRNAs targeting the highly conserved 2B gene sequence are widely used in picornavirus research, including FMDV [21,25], EMCV [23], and CVB3 [24], and significant experimental viral suppression has been achieved. Basically, RNA interference against 2B gene affects the stability and integrity of the whole viral genome.…”

“…The 2B protein is a crucial component of picornaviruses that exhibits viroporin or viroporin-like activity, plays a key role in the picornavirus life cycle by inducing a series of cytotoxic reactions to promote picornaviral replication and release [6,7,8,9,10,11,12,13,14]. The 2B protein has a highly conserved sequence, which can be exploited for viral detection [15,16,17], vaccine development [18,19,20], and RNA interference [21,22,23,24,25]. In addition, the 2B protein exhibits a viroporin or viroporin-like activity, and thus, targeted drugs against viroporin could potentially target 2B protein as a novel strategy to treat or prevent picornavirus infections.…”

RNA interference-based technology: what role in animal agriculture?

“…RNAi has been shown to inhibit a wide range of agriculturally significant pathogens both in vitro and in vivo, including foot and mouth disease virus (FMDV; Jiao et al 2013;Gismondi et al 2014), African swine fever virus (Keita et al 2010), classical swine fever virus (Porntrakulpipat et al 2010), influenza A (Stoppani et al 2015), highly pathogenic avian influenza (Stewart et al 2011), chicken anaemia virus (Hinton and Doran 2008), bovine viral diarrhoea virus (Lambeth et al 2007), and infectious bursal disease virus (Wang et al 2010).…”

Animal agriculture and the importance of agnostic governance of biotechnology

“…Without treatment, infection is almost always fatal. African swine fever pigs [47] Avian influenza chickens [48] Avian leukosis virus chickens [49] Bacteria resistance sheep/pigs/fish [50][51][52][53] Bovine spongiform encephalopathy cattle, sheep [54,55] Bovine tuberculosis cattle [56] Foot and mouth disease various [57] Grass carp haemorrhage virus (GCHV) fish [58] Influenza A & classical swine fever pigs [59] Mastitis/health of nursing offspring various [60][61][62][63][64][65] Porcine reproductive and respiratory syndrome virus (PRRSV) pigs [66] Trypanosomiasis various [30] Visna virus resistance sheep [67] Losses in livestock production due to African animal trypanosomiasis (AAT), or nagana, are estimated at US $1 billion annually [24]. Direct losses in meat production and milk yield, plus the costs for trypanosomiasis control programs, are estimated at up to US $1.2 billion each year.…”