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Foot-and-mouth-disease virus (FMDV) is a picornavirus that causes a highly contagious disease of cloven-hoofed animals resulting in economic losses worldwide. The 3C protease (3Cpro) is the main protease essential in the picornavirus life cycle, which is an attractive antiviral target. Here, we used computer-aided virtual screening to filter potential anti-FMDV agents from the natural phytochemical compound libraries. The top 23 filtered compounds were examined for anti-FMDV activities by a cell-based assay, two of which possessed antiviral effects. In the viral and post-viral entry experiments, luteolin and isoginkgetin could significantly block FMDV growth with low 50% effective concentrations (EC50). Moreover, these flavonoids could reduce the viral load as determined by RT-qPCR. However, their prophylactic activities were less effective. Both the cell-based and the fluorescence resonance energy transfer (FRET)-based protease assays confirmed that isoginkgetin was a potent FMDV 3Cpro inhibitor with a 50% inhibition concentration (IC50) of 39.03 ± 0.05 and 65.3 ± 1.7 μM, respectively, whereas luteolin was less effective. Analyses of the protein–ligand interactions revealed that both compounds fit in the substrate-binding pocket and reacted to the key enzymatic residues of the 3Cpro. Our findings suggested that luteolin and isoginkgetin are promising antiviral agents for FMDV and other picornaviruses.

Porcine circovirus type 2 (PCV2) infections may lead to the development of subclinical signs or chronic systemic syndromes, collectively known as "porcine circovirus-associated disease" (PCVAD) in swine. Interferon gamma (IFN-γ) is known to enhance PCV2 replication in vitro, and immune mediators may act as pivotal factors in triggering PCV2 infection progression toward PCVAD. We determined the effects of IFN-γ on PCV2 replication in PK-15 cells. PCV2 was cultured in the presence or absence of exogenous swine IFN-γ (swIFNγ). Growth curve analysis in PK-15 cells revealed that PCV2 could replicate to a significantly higher titer in swIFNγ medium. To investigate the host cell response upon PVC2 infection, differential expression of proteins in PCV2-infected PK-15 cells with or without swIFNγ stimulation was analyzed by proteomics (LC-MS/MS) analysis. A large proportion of the differentially expressed proteins in swIFNγ-treated PCV2-infected cells were found to be involved in apoptosis, cellular stress responses, cell survival/proliferation pathways, and inflammatory responses. We further confirmed the expression of these differentially expressed proteins at the mRNA levels by qRT-PCR. PCV2 infection in PK-15 cells in the presence of IFN-γ resulted in upregulation of cellular proteins in responses to stress, cell survival, and cell proliferation (Hsp90, MAP3K7, RAS-GTPase, c-myc, and 14-3-3 epsilon) as well as in an increase in the levels of proteins (CASP9 and TRAF5) related to the apoptosis pathways. Thus, PCV2 exploits several cellular biological processes through IFN activation for enhancing viral replication. This is the first evidence of IFN-γ promoting PCV2 replication in vitro via a mechanism similar to that used by several human viruses.

Picornaviruses are non-enveloped, single-stranded RNA viruses that cause highly contagious diseases, such as polio and hand, foot-and-mouth disease (HFMD) in human, and foot-and-mouth disease (FMD) in animals. Reverse genetics and minigenome of picornaviruses mainly depend on in vitro transcription and RNA transfection; however, this approach is inefficient due to the rapid degradation of RNA template. Although DNA-based reverse genetics systems driven by mammalian RNA polymerase I and/or II promoters display the advantage of rescuing the engineered FMDV, the enzymatic functions are restricted in the nuclear compartment. To overcome these limitations, we successfully established a novel DNA-based vector, namely pKLS3, an FMDV minigenome containing the minimum cis-acting elements of FMDV essential for intracytoplasmic transcription and translation of a foreign gene. A combination of pKLS3 minigenome and the helper plasmids yielded the efficient production of uncapped-green florescent protein (GFP) mRNA visualized in the transfected cells. We have demonstrated the application of the pKLS3 for cell-based antiviral drug screening. Not only is the DNA-based FMDV minigenome system useful for the FMDV research and development but it could be implemented for generating other picornavirus minigenomes. Additionally, the prospective applications of this viral minigenome system as a vector for DNA and mRNA vaccines are also discussed.

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