Inhibition of porcine transmissible gastroenteritis virus (TGEV) replication in mini-pigs by shRNA

Abstract

Transmissible gastroenteritis virus (TGEV) is the causative agent of porcine transmissible gastroenteritis (TGE), characterized by high mortality and severely retarded growth in piglets that dramatically affects the porcine industry. Previously, we have identified two shRNA-expressing plasmids pEGFP-U6/P1 and pEGFP-U6/P2 that target RNA-dependent RNA polymerase (RdRP) gene of TGEV with more than 95% of virus inhibition in vitro. In this study, inhibition of the TGEV replication by pEGFP-U6/P1 and pEGFP-U6/P2 was tested in mini-pigs. SPF mini-pigs at 25 days old were injected with the shRNA-expressing plasmids and then infected with TGEV. The results from the analyses of clinical signs, histopathology, indirect immunofluorescence (IIF) and RT-PCR show that the two shRNA-expressing plasmids could significantly decrease the quantity of TGEV in different organs and protect mini-pigs from TGEV infection. These findings illustrate the prospect for TGEV-specific shRNAs to be new anti-TGEV agents.

Fig. 1

shRNA-expressing plasmids protect mini-pig organs from TGEV destruction. All histology sections were stained with H&E. Panels A to D show tissue sections from mini-pigs in group 2, panels E to H show tissue sections from mini-pigs in group 5, and panels I to L show tissue sections from mini-pigs in group 1. (A) Liver with severe swelling and granular degeneration of hepatocytes (200×). (B) Kidney with serious granular degeneration of renal tubular epithelial cells, severe glomerular and renal tubular swelling (200×). (C) Jejunum with villi severe atrophied and destructed (100×). (D) Ileum with severe lymphocytes proliferation in the lamina propria (100×). (E) Liver with low-grade swelling and granular degeneration of hepatocytes, corresponding to the “+” in Table 2 (200×). (F) Kidney with normal glomeruli but low-grade renal tubular swelling, corresponding to the “+” in Table 2 (200×). (G) Jejunum with intact villi but low-grade hyperemia and lymphocytes proliferation, corresponding to the “+” in Table 2 (100×). (H) Ileum with lymphocytes proliferation in the lamina propria, corresponding to the “++” in Table 2 (100×). (I) Liver with hepatocytes in the normal state (200×). (J) Kidney with glomeruli and tubules in the normal state (200×). (K) Jejunum with villi in the normal state (100×). (L) Ileum with lamina propria in the normal state (100×).

Fig. 2

shRNA-expressing plasmids significantly reduced TGEV antigen in different organs. All sample frozen tissue sections were stained with Texas Red (original magnification, 400×). Mesenteric lymph node (A), liver (B), jejunum (C), ileum (D) and kidney (E) were sampled from the mini-pig in group 4. Red spots indicated the TGEV-infected cells. Mesenteric lymph node (F), liver (G), jejunum (H), ileum (I) and kidney (J) were sampled from the mini-pigs in group 2, served as the viral infection controls.

Fig. 3

shRNA-expressing plasmids remarkably inhibited TGEV RNA replication in different organs. (A) All the organs were sampled from the mini-pigs in group 5; (B) all the organs were sampled from the mini-pigs in group 2; M: 50 bp DNA ladder marker; A: mesenteric lymph node; B: lung; C: liver; D: jejunum; E: ileum; F: kidney; G: spleen. A_β, B_β, C_β, D_β, E_β, F_β, G_β are products of β-actin gene expressed in these organs; A_N, B_N, C_N, D_N, E_N, F_N, G_N are products of the N gene of TGEV in these organs. The TGEV N gene fragments, amplified successfully in all samples in group 2, were undetectable in group 5.