Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector

Abstract

Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.Vaccines 2019, 7, 173 2 of 12 the CV777 vaccine strain belonging to subtype G1 [11]. However, many studies demonstrated that commercially available PEDV vaccines derived from classical strains of PEDV do not provide effective protection against highly virulent PEDV variant infections in China [3]. Thus, PEDV infection remains a major veterinary problem. Understanding it is essential to developing novel antiviral drugs that will specifically inhibit PEDV propagation.RNA interference (RNAi) is a short, double-strand RNA-induced process that targets and degrades the messenger RNA (mRNA) of specific sequences [12,13]. Post-transcriptional gene silencing can be mediated by exogenous small interfering RNAs (siRNAs), endogenous microRNAs (miRNAs), and short hairpin RNAs (shRNAs). Effective gene knockdown is achieved by shRNAs by inducing the endogenous RNAi process [14,15]. Transcribed shRNAs are exported from the nucleus by Exportin-5 and processed by the RNase III Dicer into small double-stranded RNA (dsRNA) molecules of 19 to 23 bp called siRNAs. The complementary guide strand of processed siRNA is incorporated into the RNA-induced silencing complex to mediate the cleavage of target mRNAs [16,17]. RNAi evolves in the host defense system directed at infectious viruses and transposable elements. Effective silencing of transgene expression and endogenous genes in vivo was shown by a number of groups [6,7,18]. These findings raised the possibility that RNAi could be another therapeutic approach for inhibiting virus infection. ShRNAs were employed for therapy against human viral diseases, as well as cancer and neurogenerative diseases [8]. PEDV primarily infects villous epithelial cells throughout the small intestine and causes serious injury of intestine epithelial cells (IECs), including superficial villous enterocyte swelling and severe diffuse atrophic enterit...

Hypericum japonicum extract inhibited porcine epidemic diarrhea virus in vitro and in vivo

“…The invasion of pig intestinal epithelial cells by PEDV can cause enteritis in pigs of all ages and often lead to death of newborn piglets ( Wen et al, 2018 ). Although vaccines against PEDV exist, they are not optimal treatments in terms of safety and efficacy, resulting in large-scale infections still occurring ( Shi et al, 2019 ). Therefore, it is imperative to find new drugs against PEDV.…”

Current concepts in the development of therapeutics against human and animal coronavirus diseases by targeting NP

“…Two shRNAs targeting the NTD and one shRNA targeting the CTD of NPs showed dose-dependent inhibitory effects on both NP expression and PEDV replication in swine intestine epithelial cells. Particularly, all of the designed shRNAs had comparable antiviral function against the classical CV777 strain and the emerging LNCT2 strain [55] .…”

“…In addition to chemical compounds, another study revealed that RNA interference targeting PEDV NPs could be effective in inhibiting virus replication. Shi et al ., analyzed 25 sequences of PEDV NPs from different countries and designed short hairpin RNAs (shRNAs) against regions conserved between classical and emerging PEDV [55] . Cell viability was not affected by transfection of plasmids expressing the shRNAs.…”

Vaccines against RNA Viruses

“…Current vaccines against the classical CV777 strain of genotype 1 do not provide effective protection against Chinese highly virulent PEDV variant infections. Thus, Shi and colleagues [ 5 ] using a RNA interference (RNAi) approach, show that three short hairpin RNA (shRNA)-expressing plasmids directed against the viral nucleocapsid (N) present antiviral activities in intestine epithelial cells infected with a both classical CV777 and LNCT2 strains, and that these shRNAs markedly reduce viral replication of both of strains upon downregulation of N protein production; thus, these strategies, based on targeting viral processivity factors, may be feasible vaccine alternatives.…”