key: cord-0911972-qgw46e9y authors: Li, Shi‐Fang; Zhao, Fu‐Rong; Gong, Mei‐Jiao; Shao, Jun‐Jun; Xie, Yin‐Li; Chang, Hui‐Yun; Zhang, Yong‐Guang title: Antiviral activity of porcine interferon omega 7 against foot‐and‐mouth disease virus in vitro date: 2018-11-08 journal: J Med Virol DOI: 10.1002/jmv.25272 sha: e0a5f9574edcebef31eff455a8c87da227800556 doc_id: 911972 cord_uid: qgw46e9y Foot‐and‐mouth disease (FMD) is a disease of worldwide economic importance, and vaccines play an important role in preventing FMDV outbreaks. However, new control strategies are still needed since FMDV outbreaks still occur in some disease‐free countries. Currently, interferon (IFN)‐based strategies have been demonstrated to be an efficient biotherapeutic option against FMDV; however, interferon omega (IFN‐ω) has not yet been assessed in this capacity. Thus, this study evaluated the antiviral activity of porcine IFN omega 7 (PoIFN‐ω7) against FMDV. After the PoIFN‐ω7 was expressed and purified, cell proliferation assays and quantitative real‐time reverse transciption‐polymerase chain reaction were used to evaluate the effective anti‐cytopathic concentration of PoIFN‐ω7 and its effectiveness pre‐ and post‐infection with FMDV in swine kidney cells (IBRS‐2). Results showed the rHis‐PoIFN‐ω7 fusion protein was considerably expressed using Escherichia coli BL21 (DE3) strain, and the recombinant protein exhibited significant in vitro protection against FMDV, including two strains belonging to type O and A FMDV, respectively. In addition, PoIFN‐ω7 upregulated the transcription of Mx1, ISG15, OAS1, and PKR genes. These findings indicated that IFN‐ω has the potential for serving as a useful therapeutic agent to prevent FMDV or other viral outbreaks in pigs. Foot-and-mouth disease (FMD) is a highly contagious acute disease that affects cloven-hoofed domestic and wild animals, and the clinical symptoms include fever, lameness and vesicular lesions on the feet, tongue, snout, and teats. 1 Disease control includes sacrificing infected and susceptible animals, inhibiting animal movement, disinfecting contaminated premises, and vaccinating susceptible animals. 2 However, vaccination is not recommended in diseasefree-countries due to several reasons, including technical limitations in distinguishing vaccinated and infected animals, different antigenically variable strains of the virus, and trade restrictions. 3 As a result, OIE has recognized that to be effective; control measures should include vaccination in combination with the use of antiviral agents and/or immunomodulatory molecules that could rapidly control the disease before an adaptive immune response is induced. 4 Interferons (IFNs) are a group of cytokines that are divided into three types, including type I, type II and type III, and they constitute the first step in the immune response to pathogens. 5 Type I IFNs, including IFN-α, β, δ, ε, ζ, ω, κ, τ are the largest group in the interferon family and have been well-characterized for their action against viral infections. 6 IFN-ω genes were initially found in humans thirty years ago, and since then, have been identified in other animals except canines and mice. 7 Similar to other IFNs, IFN-ω is produced by cells in response to viral infection and has identical antiviral, antiproliferative, and immunomodulatory activities by binding to the same receptors and activating similar pathways to the interferon-α/β receptor. 8, 9 The feline IFN-ω was the first licensed interferon compound, which is used in cats for the treatment of feline immunodeficiency virus and feline leukemia virus infections (Virbagen®, Virbac) in Europe, Japan, Australia, New Zealand, and Mexico. 10 Unfortunately, it has been not utilized for other viral infections. In fact, IFN-ω also exerts a protective effect against several viruses, including Interestingly, different subtypes of IFN-ω have distinct antiviral activity in vitro and in vivo. For example, among the four IFN-ω pseudogenes and eight functional genes in the porcine IFN-ω (PoIFN-ω) family, porcine IFN omega 7 (PoIFN-ω7) has the highest antiviral activities, which are approximately 20 times than PoIFN-ω4, which has the lowest antiviral activity. 11 Additionally, IFN-ω has lower in vitro cytotoxicity, compared to other interferons, making it a therapeutic candidate for treating some viral diseases. 11 In this study, PoIFN-ω7 was expressed in Escherichia coli, and its antiviral effects against FMDV was assessed in vitro. The monolayers of IBRS-2 cells seeded in 12-well plates were treated with 10 ng/mL of PoIFN-ω7, and untreated cells were maintained as the control group. 24 hours post-stimulation later, cells were scraped from wells. Subsequently, total RNA was extracted using TRIzol According to the sequence of PoIFN-ω7, which was available in NCBI, the nucleotide sequences of PoIFN-ω7 were synthesized. pET-30a was used as the expression vector, which contained the sequence of one hexahistidine (his) tag fused to the N-terminus of PoIFN-ω7 16 was detected. Using a Ni-NTA agarose (IMAC) column with a buffer containing imidazole, more than 90% purity of recombinant PoIFN-ω7 protein was obtained with a yield of 5 mg/L from bacterial culture ( Figure 2A ). Subsequently, the protein was further identified by Western blot analysis ( Figure 2B ). Endotoxin was undetectable in the purified recombinant PoIFN-ω7 protein (below the detection limit, <0.01 EU/mg). To investigate the minimum protection antiviral concentration of purified protein for IBRS-2 cells, a serially diluted ten-fold from 100 ng/mL to 0.001 ng/mL of PoIFN-ω7 was added in five replicates to IBRS-2 cells. The lowest dilution of PoIFN-ω7 to offer protection to cells from CPE was 10 ng/mL, which displayed To To better illustrate the mechanism of antiviral activity elicited by Figure 3C ). In fact, unlike other vaccines, IFNs exert their antiviral activity against lots of viruses, no matter which the serotype or strain infected. [23] [24] [25] This result was similar to the study from Usharani et al, 22 in their study, they found that IFN-τ4 also exerted broad-spectrum antiviral effects against eight strains of FMDV. In post-infection study, PoIFN-ω7 (10 ng/mL) also provided cells considerable protection against CPE and displayed 1.29-log reduction in viral mRNA levels at 0-8 hours post-infection, compared to the VC group ( Figure 4 ). Viral mRNA levels increased at 0 to 8 hours post-infection and raised considerably after 8 hours, while no considerable CPE was observed in this period, these results suggest that PoIFN-ω7 might serve as an useful adjunct treatment at the onset of the FMD outbreak as well as that within 8 hours of exposure to FMDV. Generally, type I IFNs trigger the production of antiviral effectors by mediating the JAK/STAT pathway, these effectors include ISG, Mx1, OAS, and PKR. 26 In recent years, there has been increasing interest in studying the consensus interferon (a type IFN that contain the most frequently occurring amino acids present among the non-allelic subtypes), which has been shown to have higher antiviral and anti-proliferative activities, natural killer cell activation and ISG-induction activities, compared to single IFN subtypes. 29 Likewise, porcine IFN-ω is a multigenic family containing seven subtypes with different antiviral activities and different expression profiles. 30 Therefore, a consensus IFN-ω could also be designed, and its efficacy against FMDV should be investigated in the future studies. In conclusion, PoIFN-ω7 exhibited in vitro antiviral activities against FMDV and provides new insights for developing a novel The authors declare no conflicts of interest. 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