Inhibition of porcine transmissible gastroenteritis virus infection in porcine kidney cells using short hairpin RNAs targeting the membrane gene

Abstract

The membrane (M) protein is the most abundant component of the porcine transmissible gastroenteritis virus (TGEV) particle. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, three plasmids (pRNAT-1, pRNAT-2, and pRNAT-3) expressing short hairpin RNAs were designed to target three different coding regions of the M gene of TGEV. The plasmids were constructed and transiently transfected into a porcine kidney cells, PK-15, to determine whether these constructs inhibited TGEV production. The analysis of cytopathic effects demonstrated that pRNAT-2 and pRNAT-3 could protect PK-15 cells against pathological changes specifically and efficiently. Additionally, indirect immunofluorescence and 50% tissue culture infectious dose (TCID) assays showed that pRNAT-2 and pRNAT-3 inhibited the multiplication of the virus at the protein level effectively. Quantitative real-time PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the three plasmids were reduced by 13, 68, and 70%, respectively. This is the first report showing that RNAi targeting of the M gene. Our results could promote studies of the specific function of viral genes associated with TGEV infection and might provide a theoretical basis for potential therapeutic applications.

Short hairpin RNAs targeting M and N genes reduce replication of porcine deltacoronavirus in ST cells

“…The inhibitory efficiency of pGenesil-N was higher than that of pGenesil-M. Although the interference efficiency of shRNAs against PDCoV in our study was lower than that of the shRNAs targeting other coronaviruses [15,17], these results indicate that RNAi against PDCoV mediated by shRNAs can inhibit PDCoV replication in vitro. A disadvantage of this transient transfection/expression system is that virus replication can be inhibited only in cells that are expressing the shRNA, and cells not expressing the shRNA can be infected.…”

“…Therefore, RNAi has been considered as an effective strategy to protect against bacterial and viral pathogens [22,23]. RNAi is triggered by endogenous or exogenous 21-23 nt RNA duplexes [17], and shRNA and siRNA are two commonly used RNA molecules to block gene expression [17,24]. Compared with siRNA, the interference efficiency induced by shRNA was more effective.…”

“…Wang designed and constructed three recombinant plasmids targeting the M gene of porcine TGEV. After transfection into PK15 cells, M gene expression was reduced by 13, 68, and 70% [17]. Shen et al [15] have constructed five shRNA-expressing plasmids targeting the N, M, and S genes of PEDV.…”

“…selectively inactivates the mRNA of the target gene. RNAi has been successfully used in infection inhibition studies of animal viruses, such as porcine epidemic diarrhea virus (PEDV) [15], influenza virus A [16], porcine transmissible gastroenteritis virus (TGEV) [17], and porcine reproductive and respiratory syndrome virus [18]. However, whether RNAi inhibits the replication of PDCoV has not been reported.…”

Relationship of Noncoding RNA and Swine Viruses

“…So far, RNAi is being increasingly used to inhibit the replication of swine virus such as porcine TGEV [85], foot-and-mouth disease virus [86], and PRRSV [87][88][89]. As the therapy is considered as simpler and easier to im- plement, an ideal RNAi treatment for viruses will exhibit a more competitive advantage than the use of antiviral drugs [84].…”