Porcine interferon-α linked to the porcine IgG-Fc induces prolonged and broad-spectrum antiviral effects against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an economically important disease, and the FMD virus (FMDV) can spread rapidly in susceptible animals. FMD is usually controlled through vaccination. However, commercial FMD vaccines are only effective 4-7 days after vaccination. Furthermore, FMDV comprises seven serotypes and various topotypes, and these aspects should be considered when selecting a vaccine. Antiviral agents could provide rapid and broad protection against FMDV. Therefore, this study aimed to develop a fusion protein of consensus porcine interferon-α and Fc portion of porcine antibody IgG (poIFN-α-Fc) using a baculovirus expression system to develop a novel antiviral agent against FMDV. We measured the antiviral effects of the poIFN-α-Fc protein against FMDV and the enhanced duration in vitro and in vivo. The broad-spectrum antiviral effects were tested against seven FMDV serotypes, porcine reproductive and respiratory syndrome virus (PRRSV), and bovine enterovirus (BEV). Furthermore, the early protective effects and neutralizing antibody levels were tested by co-injecting poIFN-α-Fc and an FMD-inactivated vaccine into mice or pigs. Sustained antiviral effects in pig sera and mice were observed, and pigs injected with a combination of the poIFN-α-Fc and an inactivated FMD vaccine were protected against FMDV in a dose-dependent manner at 2- and 4-days post-vaccination. In addition, combined with the inactivated FMD vaccine, poIFN-α-Fc increased the neutralizing antibody levels in mice. Therefore, poIFN-α-Fc is a potential broad-spectrum antiviral and adjuvant candidate that can be used with inactivated FMD vaccines to protect pigs against FMDV.

Interferon alpha and beta receptor 1 knockout in human embryonic kidney 293 cells enhances the production efficiency of proteins or adenoviral vectors related to type I interferons.

Human embryonic kidney (HEK) 293 cells are widely used in protein and viral vector production owing to their high transfection efficiency, rapid growth, and suspension growth capability. Given their antiviral, anticancer, and immune-enhancing effects, type I interferons (IFNs) have been used to prevent and treat human and animal diseases. However, the binding of type I IFNs to the IFN-α and-ß receptor (IFNAR) stimulates the expression of IFN-stimulated genes (ISGs). This phenomenon induces an antiviral state and promotes apoptosis in cells, thereby impeding protein or viral vector production. In this study, we generated an IFNAR subtype 1 knockout (KO) HEK 293 suspension (IFNAR-KO) cell line by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology. Upon treatment with human IFN-α, the IFNAR-KO cells showed a constant expression of ISGs, including 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (Mx1), protein kinase RNA-activated (PKR), and IFN-induced transmembrane protein 1 (IFITM1), when compared with the wild-type HEK 293 (WT) cells, wherein the ISGs were significantly upregulated. As a result, the titer of recombinant adenovirus expressing porcine IFN-α was significantly higher in the IFNAR-KO cells than in the WT cells. Furthermore, the IFNAR-KO cells continuously produced higher amounts of IFN-α protein than the WT cells. Thus, the CRISPR-Cas9-mediated IFNAR1 KO cell line can improve the production efficiency of proteins or viral vectors related to IFNs. The novel cell line may be used for producing vaccines and elucidating the type I IFN signaling pathway in cells.

Preventive effects of quercetin against foot-and-mouth disease virus in vitro and in vivo by inducing type I interferon.

Foot-and-mouth disease (FMD) is an acute contagious infectious disease that affects cloven-hoofed animals. Although current emergency FMD vaccines only take effect 7 days after vaccination, antiviral agents, such as quercetin, which is a common flavonoid, could reduce the spread of FMD virus (FMDV) during outbreaks. We investigated the in vitro and in vivo antiviral effects of quercetin against FMDV. Analysis of viral copy numbers showed that quercetin had a dose-dependent inhibitory effect on FMDV at concentrations between 19.5 and 1,250 µM in porcine cells. In addition, we observed a quercetin-induced interferon (IFN)-α protein and interferon-stimulated gene (ISG) upregulation in swine cells. Enzyme-linked immunosorbent assay of sera revealed that quercetin induces the production of IFN-α, IFN-ß, IFN-γ, interleukin (IL)-12, and IL-15 in mice. Inoculation of mice with quercetin or a combination of quercetin with an inactivated FMD vaccine enhanced both the survival rate and neutralizing antibody titer. Therefore, we suggest the use of quercetin as a novel and effective antiviral agent for controlling FMDV infection; however, further investigation of its application in livestock is required.

Antiviral effect of vesatolimod (GS-9620) against foot-and-mouth disease virus both in vitro and invivo.

Foot-and-mouth disease (FMD) is an acute contagious disease of cloven-hoofed animals such as cows, pigs, sheep, and deer. The current emergency FMD vaccines, to induce early protection, have limited use, as their protective effect in pigs does not begin until 7 days after vaccination. Therefore, the use of antiviral agents would be required for reducing the spread of foot-and-mouth disease virus (FMDV) during outbreaks. Vesatolimod (GS-9620), a toll-like receptor 7 agonist, is an antiviral agent against various human disease-causing viruses. However, its antiviral effect against FMDV has not been reported yet. The aim of this study was to investigate the antiviral effects of GS-9620 against FMDV both in vitro and in vivo. The inhibitory effect of GS-9620 on FMDV in swine cells involved the induction of porcine interferon (IFN)-α and upregulation of interferon-simulated genes. Protective effect in mice injected with GS-9620 against FMDV was maintained for 5 days after injection, and cytokines such as IFN-γ, interleukin (IL)-12, IL-6, and IFN-γ inducible protein-10 could be detected following the treatment with GS-9620. Furthermore, the combination of GS-9620 with an FMD-inactivated vaccine was found to be highly effective for early protection in mice. Overall, we suggest GS-9620 as a novel and effective antiviral agent for controlling FMDV infection.

BacMam Expressing Highly Glycosylated Porcine Interferon Alpha Induces Robust Antiviral and Adjuvant Effects against Foot-and-Mouth Disease Virus in Pigs.

Foot-and-mouth disease (FMD) is an acute contagious disease that affects cloven-hoofed animals and has severe global economic consequences. FMD is most commonly controlled by vaccination. Currently available commercial FMD vaccines contain chemically inactivated whole viruses, which are thought to be slow acting as they are effective only 4 to 7 days following vaccination. Hence, the development of a novel rapid vaccine or alternative measures, such as antiviral agents or the combination of vaccines and antiviral agents for prompt FMD virus (FMDV) outbreak containment, is desirable. Here, we constructed a recombinant baculovirus (BacMam) expressing consensus porcine interferon alpha (IFN-α) that has three additional N-glycosylation sites driven by a cytomegalovirus immediate early (CMV-IE) promoter (Bac-Con3N IFN-α) for protein expression in mammalian cells. Bac-Con3N IFN-α expressing highly glycosylated porcine IFN-α protein increased the duration of antiviral effects. We evaluated the antiviral effects of Bac-Con3N IFN-α in swine cells and mice and observed sustained antiviral effects in pig serum; additionally, Bac-Con3N IFN-α exhibited sustained antiviral effects in vivo as well as adjuvant effects in combination with an inactivated FMD vaccine. Pigs injected with a combination of Bac-Con3N IFN-α and the inactivated FMD vaccine were protected against FMDV at 1, 3, and 7 days postvaccination. Furthermore, we observed that in combination with the inactivated FMD vaccine, Bac-Con3N IFN-α increased neutralizing antibody levels in mice and pigs. Therefore, we suggest that Bac-Con3N IFN-α is a strong potential antiviral and adjuvant candidate for use in combination with inactivated FMD vaccines to protect pigs against FMDV. IMPORTANCE Early inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in pigs is highly desirable as FMDV transmission and shedding rates are higher in pigs than in cattle. However, commercial FMD vaccines require at least 4 to 7 days postvaccination (dpv) for protection, and animals are vulnerable to heterologous viruses before acquiring high antibody levels after the second vaccination. Therefore, the development of antiviral agents for use in combination with FMD vaccines is essential. We developed a novel antiviral and immunostimulant, Bac-Con3N IFN-α, which is a modified porcine IFN-α-expressing recombinant baculovirus, to improve IFN stability and allow its direct delivery to animals. We present a promising candidate for use in combination with inactivated FMD vaccines as pigs applied to the strategy had early protection against FMDV at 1 to 7 dpv, and their neutralizing antibody levels were higher than those in pigs administered the vaccine only.

A Vaccine Strain of the A/ASIA/Sea-97 Lineage of Foot-and-Mouth Disease Virus with a Single Amino Acid Substitution in the P1 Region That Is Adapted to Suspension Culture Provides High Immunogenicity.

There are seven viral serotypes of foot-and-mouth disease virus (FMDV): A, O, C, Asia 1, and Southern African Territories 1, 2, and 3 (SAT 1-3). Unlike serotype O FMDV vaccine strains, vaccine strains of serotype A FMDV do not provide broad-range cross-reactivity in serological matching tests with field isolates. Therefore, the topotype/lineage vaccine strain circulating in many countries and a highly immunogenic strain might be advantageous to control serotype A FMDV. We developed a new vaccine strain, A/SKR/Yeoncheon/2017 (A-1), which belongs to the A/ASIA/Sea-97 lineage that frequently occurs in Asian countries. Using virus plaque purification, we selected a vaccine virus with high antigen productivity and the lowest numbers of P1 mutations among cell-adapted virus populations. The A/SKR/Yeoncheon/2017 (A-1) vaccine strain has a single amino acid mutation, VP2 E82K, in the P1 region, and it is perfectly adapted to suspension culture. The A/SKR/Yeoncheon/2017 (A-1) experimental vaccine conferred high immunogenicity in pigs. The vaccine strain was serologically matched with various field isolates in two-dimensional virus neutralization tests using bovine serum. Vaccinated mice were protected against an A/MAY/97 virus that was serologically mismatched with the vaccine strain. Thus, A/SKR/Yeoncheon/2017 (A-1) might be a promising vaccine candidate for protection against the emerging FMDV serotype A in Asia.

Analysis of Amino Acid Mutations of the Foot-and-Mouth Disease Virus Serotype O Using both Heparan Sulfate and JMJD6 Receptors.

Foot-and-mouth disease (FMD) is an economically devastating animal disease. Adapting the field virus to cells is critical to the vaccine production of FMD viruses (FMDV), and heparan sulfate (HS) and Jumonji C-domain-containing protein 6 (JMJD6) are alternative receptors of cell-adapted FMDV. We performed serial passages of FMDV O/SKR/Andong/2010, classified as the O/Mya-98 topotype/lineage and known as a highly virulent strain, to develop a vaccine seed virus. We traced changes in the amino acid sequences of the P1 region, plaque phenotypes, and the receptor usage of the viruses, and then structurally analyzed the mutations. VP3 H56R and D60G mutations were observed in viruses using the HS receptor and led to changes in the hydrogen bonding between VP3 56 and 60. A VP1 P208L mutation was observed in the virus using the JMJD6 receptor during cell adaptation, enabling the interaction with JMJD6 through the formation of a new hydrogen bond with JMJD6 residue 300. Furthermore, VP1 208 was near the VP1 95/96 amino acids, previously reported as critical mutations for JMJD6 receptor interactions. Thus, the mutation at VP1 208 could be critical for cell adaptation related to the JMJD6 receptor and may serve as a basis for mechanism studies on FMDV cell adaptation.