Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo

Abstract

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.

FIG. 1.

Schematic diagram of target viral mRNA, shRNA-expressing cassette, Ad5 shuttle vector, and rAd5 DNA. (A) The FMDV genome contains a unique open reading frame. The black arrows underneath indicate the sites targeted by FMDV-specific shRNAs. (B) An inverted repeat corresponding to each of the target sequences in the FMDV genome was inserted under the control of P_U6 and a transcriptional termination signal of five Ts. The shRNA-expressing cassette was then subcloned into the multiple cloning sites of Ad5 shuttle vector pAdTrack-CMV under the control of P_CMV and a poly(A) transcription termination signal (An). As a result, transcription of the shRNA-coding insert could be driven by either P_U6 or P_CMV. The synthesized RNAs should therefore fold back to form two types of shRNAs that are finally processed into the putative siRNAs. (C) The resultant Ad5 shuttle vector was cotransfected with the adenoviral backbone plasmid pAdEasy-1 into E. coli by electroporation. The recombinant adenoviral DNAs were generated by homologous recombination.

FIG. 2.

rAd5 expressing FMDV-specific shRNA confers specific resistance to FMDV in IBRS-2 cells. Cells treated with either Ad5-LacZ or FMDV-specific rAd5 at an MOI of 5 were challenged, 12 h posttreatment, with 100 TCID₅₀ of FMDV HKN/2002 (A), FMDV CHA/99 (B), or PRV Ea (C). At 72 h after challenge, the cells were observed with an Olympus BH-2 microscope, and representative bright-field images (left column) and relative fluorescent-field images (right column) were recorded.

FIG. 3.

rAd5 expressing FMDV-specific shRNA protects IBRS-2 cells from virus infection. Cells were inoculated with shRNA-expressing rAd5 at an MOI of 5 and challenged, 12 h postinoculation, with 100 TCID₅₀ of FMDV HKN/2002 (A), FMDV CHA/99 (B), or PRV Ea (C). Culture supernatants were collected at several times after FMDV challenge, and virus yields were measured by TCID₅₀. The data are means ± the standard deviation of three separate experiments.

FIG. 4.

The level of FMDV 3D transcripts in IBRS-2 cells treated with FMDV-specific rAd5. Cells were inoculated with shRNA-expressing rAd5 at an MOI of 5 and challenged 12 h postinoculation with 100 TCID₅₀ of FMDV HKN/2002. At several hours postchallenge (h.p.c.), total RNA was extracted from cultures and subjected to real-time Q-RT-PCR. (A) One amplification plot of two separate experiments is shown. The y axis represents the PCR baseline-subtracted RFU (relative fluorescence units). Cycle number is displayed on the x axis. (B) Cycle threshold (C_T) values are derived from the amplification profiles shown in panel A.

FIG. 5.

Antiviral activity of shRNA-expressing rAd5 in swine. Animals were treated with PBS (A), Ad5-LacZ (B), Ad5-NT21-Ad5-POL mixture (C), or a high dose of Ad5-NT21-Ad5-POL mixture (D) and challenged as described in Materials and Methods. After challenge, lesion scores were assigned to the animals according to the number of digits plus mouth with vesicles.