Evaluation of a combinatorial RNAi lentivirus vector targeting foot-and-mouth disease virus in vitro and in vivo

Abstract

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven‑hoofed animals, which leads to serious economical losses. FMDV is not adequately controlled by vaccination or biosecurity measures. To generate genetically modified FMDV‑resistant animals, a combinatorial expression cassette producing three short hairpin (sh)RNAs was constructed using the lentivirus (LV) vector, LV‑3shRNA. The three shRNAs were expressed under the regulation of DNA polymerase III promoters from a buffalo and a bovine source, with one targeted to the non‑structural protein 3B, and the other two targeted to the viral polymerase protein 3D of FMDV, respectively. The role of LV‑3shRNA in the inhibition of the replication of FMDV was determined in BHK‑21 cells and in suckling mice. The results revealed that LV‑3shRNA reduced viral growth 3‑fold (24 h post‑infection) when the cells were challenged with 107‑times the tissue culture infective dose (TCID50)/ml of O serotype FMDV. The suckling mice pretreated with LV‑3shRNA were completely protected on administration of 5‑times the dose of FMDV otherwise sufficient to kill 50% of the experimental animals (LD50). These results demonstrated that the LV‑mediated dual expression of three FMDV‑specific shRNAs provided a novel strategy towards combating FMDV, which facilitates the permanent introduction of novel disease-resistance traits into the buffalo and bovine genomes in the future.

Figure 1

FMDV-specific shRNA sequences and multi-shRNA expression construct design. (A) The sequences of three FMDV-specific shRNAs (Cons1, Cons2 and Cons3) were selected from three fragments located in the 3B or 3D protein coding regions, respectively, which are illustrated featuring the sense, loop, antisense and DNA polymerase III terminator (TTTTTT) sequences. (B) The alignment of several strains of the A, Asia I, C and O serotype FMDV genome are shown. The sequences indicated by different colors represent the conserved sites. (C) The design of the multi-shRNA expression construct in the lentiviral plasmid pSicoR is illustrated schematically. boU6, bovine U6 promoter; bu7SK, buffalo 7SK promoter; buU6, buffalo U6 promoter; Cons, constant; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; FMDV, foot-and-mouth disease virus; HIV-1 5′-LTR, human immunodeficiency virus-1 5′-long terminal repeat; RRE, HIV-1 Rev response element; shRNA, short hairpin RNA; UTR, untranslated region; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2

Detection of exogenous gene expression in BHK-21-LV transgenic cells. (A) Fluorescence microscopy images (scale bar, 400 µm) of the BHK-LV cells transfected with the lentiviral vector, LV-3shRNA, are shown. (B) The transcriptional efficiency of different shRNAs in the BHK-21-LV and normal BHK-21 cells were quantified by reverse transcription-quantitative polymerase chain reaction. The data are presented as the percentage of the expression of Cons3 in the BHK-21-LV cells as the mean ± standard error of the mean of three replicates. Cons, construct; shRNA, short hairpin RNA; LV, lentivirus.

Figure 3

An application curve of FMDV in the BHK-21-LV-treated cells by reverse transcription-quantitative polymerase chain reaction. The filled-in circles represent FMDV RNA copies in the BHK-21-LV cells at 6–48 h post-infection, and the filled-in squares represent the viral RNA copies in the negative control BHK-21 cells. Error bars represent the standard error of the mean calculated from at least three independent experiments. FMDV, foot-and-mouth disease virus; LV, lentivirus.

Figure 4

Mortality data for suckling mice pretreated with LV-3shRNA and challenged with FMDV. The LV group of suckling mice were treated with LV-3shRNA 72 h prior to viral infection, whereas the NC group was treated with normal saline solution. Each group of suckling mice were infected with FMDV O/HKN/2002 on day 0. The survival of the mice is indicated by the length of the blue bar. The terminal block color indicates the day and the cause of death (black, found dead; purple, pathogenetic; orange, moribund; yellow, healthy mice sacrificed for immunohistological studies). The numbers shown are the log₁₀ of the number of viral RNA copies present in carcass samples 72 h post-infection. FMDV, foot-and-mouth virus; LD₅₀, median lethal dose; LV, lentivirus; NC, negative control.