Robust Protection against Highly Virulent Foot-and-Mouth Disease Virus in Swine by Combination Treatment with Recombinant Adenoviruses Expressing Porcine Alpha and Gamma Interferons and Multiple Small Interfering RNAs.

UNLABELLED: Because the currently available vaccines against foot-and-mouth disease (FMD) provide no protection until 4 to 7 days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is the application of antiviral agents. Combination treatment strategies have been used to enhance the efficacy of antiviral agents, and such strategies may be advantageous in overcoming viral mechanisms of resistance to antiviral treatments. We have developed recombinant adenoviruses (Ads) for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) targeting FMDV mRNAs encoding nonstructural proteins. The antiviral effects of Ad-porcine IFN-αγ and Ad-3siRNA expression were tested in combination in porcine cells, suckling mice, and swine. We observed enhanced antiviral effects in porcine cells and mice as well as robust protection against the highly pathogenic strain O/Andong/SKR/2010 and increased expression of cytokines in swine following combination treatment. In addition, we showed that combination treatment was effective against all serotypes of FMDV. Therefore, we suggest that the combined treatment with Ad-porcine IFN-αγ and Ad-3siRNA may offer fast-acting antiviral protection and be used with a vaccine during the period that the vaccine does not provide protection against FMD. IMPORTANCE: The use of current foot-and-mouth disease (FMD) vaccines to induce rapid protection provides limited effectiveness because the protection does not become effective until a minimum of 4 days after vaccination. Therefore, during outbreaks antiviral agents remain the only available treatment to confer rapid protection and reduce the spread of foot-and-mouth disease virus (FMDV) in livestock until vaccine-induced protective immunity can become effective. Interferons (IFNs) and small interfering RNAs (siRNAs) have been reported to be effective antiviral agents against FMDV, although the virus has associated mechanisms of resistance to type I interferons and siRNAs. We have developed recombinant adenoviruses for the simultaneous expression of porcine alpha and gamma interferons (Ad-porcine IFN-αγ) as well as 3 small interfering RNAs (Ad-3siRNA) to enhance the inhibitory effects of these antiviral agents observed in previous studies. Here, we show enhanced antiviral effects against FMDV by combination treatment with Ad-porcine IFN-αγ and Ad-3siRNA to overcome the mechanisms of resistance of FMDV in swine.

A recombinant adenovirus bicistronically expressing porcine interferon-α and interferon-γ enhances antiviral effects against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a virulent and economically costly disease in domestic livestock. Since the current vaccine available against FMD provides no protection until 7days postvaccination, the only alternative method to halt the spread of the FMD virus (FMDV) during outbreaks is by the application of anti-viral agents. The combination of recombinant adenovirus expressing type I interferon (IFN-α) and adenovirus expressing type II IFN (IFN-γ) has been reported to be an effective anti-viral treatment strategy against FMDV. Nevertheless, the recombinant adenovirus mixture may be inefficient because of the low anti-viral efficiency of IFN-γ compared to that of IFN-α. In this study, we generated a recombinant adenovirus co-expressing porcine IFN-α and IFN-γ in tandem using an FMDV 2A sequence to mediate effective cleavage of the two proteins (referred to as Ad-porcine IFN-αγ). We demonstrated that both recombinant porcine IFN-α and IFN-γ were expressed and interferon stimulated gene (ISG)s related with IFN-α and IFN-γ were induced in porcine kidney (IBRS-2) cells infected with Ad-porcine IFN-αγ. Additionally, the anti-viral effects of Ad-porcine IFN-αγ against FMDV were enhanced both in IBRS-2 cells and in CD-1 (ICR) suckling mice compared to that of adenovirus expressing only a single protein. We propose that Ad-porcine IFN-αγ could be a rapid, highly efficient, convenient anti-viral agent against FMDV.

Enhanced inhibition of foot-and-mouth disease virus by combinations of porcine interferon-α and antiviral agents.

Foot-and-mouth disease (FMD) is an economically significant animal disease because of the speed of its transmission. The current FMD vaccine provides no protection until 7days after the vaccination, which reduces its effectiveness in the case of an outbreak. Therefore, to find an alternative method of applying antiviral agents for rapid and enhanced inhibition of the FMD virus (FMDV), we compared the antiviral effects of promising antiviral agents and attempted to apply them in combination. First, we measured and compared the 50% effective concentration (EC(50)) to the mean inhibition effects of FMDV, and the 50% cytotoxic concentration (CC(50)) to the mean cytotoxicity of antiviral agents such as ribavirin, guanidine-hydrochloride (guanidine-HCl), 6-azauridine, and recombinant adenovirus expressing three small interference RNAs (Ad-siRNA) or porcine interferon-α (Ad-porcine IFN-α) in swine kidney cells (IBRS-2). The selectivity indices of ribavirin (35.2) and 6-azauridine (34.6) were higher than that of guanidine-HCl (26.9). The selectivity indices of Ad-siRNA or Ad-porcine IFN-α were 7×10(3) or 7×10(4) based on the adenoviral titer. Next, we tested the combined effects of the FMDV inhibition agents. Enhanced inhibition effects were observed in the IBRS-2 cells and in suckling mice from the combination of Ad-porcine IFN-α and Ad-siRNA or ribavirin. The combined application of these recombinant adenoviruses and ribavirin may enhance their inhibitory effect on FMDV and overcome FMDV resistance against antiviral agents.