Enhanced antiviral activity against foot-and-mouth disease virus by a combination of type I and II porcine interferons

Abstract

Previously, we showed that type I interferon (alpha/beta interferon [IFN-alpha/beta]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 10(9) PFU of human adenovirus type 5 expressing porcine IFN-alpha (Ad5-pIFN-alpha) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-gamma) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-alpha, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 10(8) PFU of Ad5-pIFN-alpha, low- or high-dose Ad5-pIFN-gamma, or a combination of Ad5-pIFN-alpha and low- or high-dose Ad5-pIFN-gamma and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-alpha or a low dose of Ad5-pIFN-gamma developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-gamma was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-gamma or with only high-dose Ad5-pIFN-gamma were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.

FIG. 1.

Neutralization of IFN activity by MAbs. MAbs K9 against pIFN-α (a-pIFN-α) and P2C11 against pIFN-γ (a-pIFN-γ) as well as normal mouse serum (NMS) were diluted 1:500 and incubated individually or together for 1 h at RT with 1 unit of pIFN-α and 1 unit of pIFN-γ. Treated or untreated pIFNs were incubated with IBRS-2 cells for 24 h and infected with approximately 100 plaques of FMDV. Plaques were detected by crystal violet staining.

FIG. 2.

Effect of pIFN-α and pIFN-γ on the yield of FMDV A12 in IBRS-2 cells. (A) Cells were pretreated for 24 h with various amounts of pIFN-α or pIFN-γ and 24 h later infected with FMDV. After a 1-h adsorption, the cells were rinsed with 150 mM NaCl-20 mM MES (pH 6) and with MEM. Supernatants were collected at 1 and 24 h p.i. and titrated on BHK-21 cells. The results are expressed as the virus titer (number of PFU per ml) at 24 h p.i. after subtracting the titers at 1 h p.i. (B) Cells were pretreated with 1 or 2 units of pIFN-α plus increasing amounts of pIFN-γ and 24 h later infected with FMDV as described above. The results are expressed as the virus titer (number of PFU per ml) at 24 h p.i. after subtraction of the titers at 1 h p.i.

FIG. 3.

RIP of FMDV A24-infected cell lysates with 21-dpc swine sera. [³⁵S]methionine-labeled cell lysates from FMDV A24-infected IBRS-2 cells were immunoprecipitated with 21-dpc swine sera. Lane 1, bovine convalescent-phase serum; lane 2, 0-dpc serum from swine 69; lanes 3 to 20, 21-dpc serum from swine 62 to 79 in the groups indicated in the figure. Immunoprecipitated samples were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ctl, control; Comb., combination.