Antiviral Effects of Antisense Morpholino Oligomers in Murine Coronavirus Infection Models

Evaluation of PMO sequences and various peptide conjugates in MHV-infected cells. (A) Dose-dependent inhibition of MHV plaque expansion. DBT cell cultures were inoculated with fixed doses of MHV-A59, and an R₉F₂-PMO was added with each agarose overlay. The plaque diameter was then measured 72 h after the overlay was applied. (B) R₉F₂-PMO treatment reduces syncytium formation by MHV-1 and MHV-4. Qualitative changes in cell morphology and density were compared against untreated, uninfected (left) and untreated, infected (right [column marked untreated]) controls. Representative images show cells pretreated for 3 h with 10 μM R₉F₂-PMO and fixed for 24 h after inoculation. (C) Effects of delivery peptide conjugation on PMO activity. DBT cells were treated with 20 μM P-PMO for 3 h before inoculation at a multiplicity of 0.1 PFU/cell with MHV-A59. The virus yield was quantified 24 h after inoculation. 5′-Terminal peptide conjugates follow standard single-letter amino acid naming, except as follows: X, 6-aminohexanoic acid; B, beta-alanine; and Man, mannose. Asterisks indicate significant differences (P < 0.05 by Student's t test) with respect to mock-treated controls. (D) Dose-response experiment comparing the effects of R₉F₂- and (RXR)₄-conjugated 5TERM, TRS1, and RND P-PMOs on viral titers. Cells were treated with various P-PMOs at 10 μM for 3 h, infected with MHV-A59 for 1 h, and then incubated again in the presence of P-PMOs for 24 h. Error bars throughout indicate standard errors of the means.

Single-dose and long-term inhibition of coronavirus multiplication. (A) Inhibition of replication of various MHV strains (inoculated at multiplicities of 0.01 to 1 PFU/cell) in DBT cells after a 3-h preinoculation treatment with 10 μM P-PMO. The virus yield was quantified 24 h after inoculation. (B) Dose-activity comparison of the effects of SARS-5TERM and SARS-TRS1 P-PMOs on SARS-CoV (1 PFU/cell) in Vero-E6 cells. Cells were treated with P-PMOs for 6 h prior to inoculation, and viral titers were analyzed 48 h later. (C) Effects of long-term P-PMO treatment on MHV replication. DBT cells were treated for 6 h with P-PMOs and then inoculated with plaque-purified MHV-A59 (0.1 PFU/cell). Culture medium was used to inoculate fresh cultures of P-PMO-treated cells every 24 h for a total of 10 viral passages. The virus yield was quantified as described for panel A. Error bars throughout indicate standard errors of the means.

5TERM PMO and P-PMO reduce viral titers in the livers of MHV-infected mice. Mice were inoculated i.p. with MHV-A59 (A and B) or various other MHV strains (C). Starting 5 h before infection and on each of the next 2 days, each animal received one dose of saline (black bars), RND PMO or (RXR)₄-PMO (hatched bars), or 5TERM PMO or (RXR)₄-PMO (white bars), as indicated in Materials and Methods. (RXR)₄-PMO was utilized for panels B and C. Unc, unconjugated. Livers were collected on day 4 (A and C) or at the indicated time points (B), and viral titers were determined by plaque assay. Error bars indicate standard errors of the means. Asterisks indicate significant differences from mock-treated controls (P < 0.05 by Student's t test).

5TERM-(RXR)₄-PMO attenuates viral hepatitis. (A) Livers were obtained from mice infected with the indicated MHV strains at 4 (MHV-2, MHV-3, and MHV-Alb139) or 6 (MHV-A59) days p.i. The numbers of necrotic lesions per unit area were compared between P-PMO-treated and mock-treated groups, as described in Materials and Methods. Data points represent individual mice, and bars indicate the mean severities for the combined mock- and RND P-PMO-treated groups and the 5TERM P-PMO-treated group, respectively. The number of lesions was significantly lower in A59-infected mice treated with 5TERM P-PMO than in mock-treated (P < 0.05) or RND P-PMO-treated (P < 0.005) controls. (B to G) Representative areas of H&E-stained livers from uninfected (B and C) and MHV-A59 (D and E [6 days p.i.])- and MHV-3 (F and G [4 days p.i.])-infected animals. Mice received (RXR)₄-5TERM P-PMO treatment (C, E, and G) or mock treatment (B, D, and F). Necrotic lesions and inflammation characteristic of lymphocytes are indicated with arrowheads. Note the regions of bright pink staining that mark necrotic areas in panel E. (H) At a higher magnification, the liver sections show ballooning necrosis (large black arrowheads), lymphocytic infiltration with more moderate necrosis (large white arrowheads), widespread coagulation necrosis with karyorrhectic debris (small black arrowheads), and eosinophilic staining reminiscent of Councilman bodies (small white arrowheads). Bars, 0.1 mm.

Dual effects of (RXR)₄-5TERM on weight loss and survival. Mice were infected with 10 PFU MHV-3 (A) or 100 (B), 10,000 (C), or 50,000 (D) PFU MHV-Alb139. Depending on the experiment, some animals received (RXR)₄-5TERM, following either a prophylactic (empty circles) or therapeutic (empty squares and empty triangles) regimen. Controls received either saline (black circles) or (RXR)₄-RND (black triangles) daily, with the first injection starting 5 h before inoculation. The relative weight is shown for each treatment group (n = 3 to 7). Error bars indicate standard errors of the means. Each death is represented with the group's corresponding symbol positioned on the time axis at the time of the event. Asterisks indicate significant differences relative to the mock-treated control group. *, P < 0.05; **, P < 0.01. Student's t test (weight) or the log rank survival test (mortality) was used to determine statistical significance.