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 (TCID50) 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.