Foot-and-mouth Disease Virus Infection Research Articles

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication

ABSTRACT Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host’s antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-β promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection. Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4’,6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-β:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2

A genome-wide CRISPR screening uncovers that TOB1 acts as a key host factor for FMDV infection via both IFN and EGFR mediated pathways.

The interaction between foot-and-mouth disease virus (FMDV) and the host is extremely important for virus infection, but there are few researches on it, which is not conducive to vaccine development and FMD control. In this study, we designed a porcine genome-scale CRISPR/Cas9 knockout library containing 93,859 single guide RNAs targeting 16,886 protein-coding genes, 25 long ncRNAs, and 463 microRNAs. Using this library, several previously unreported genes required for FMDV infection are highly enriched post-FMDV selection in IBRS-2 cells. Follow-up studies confirmed the dependency of FMDV on these genes, and we identified a functional role for one of the FMDV-related host genes: TOB1 (Transducer of ERBB2.1). TOB1-knockout significantly inhibits FMDV infection by positively regulating the expression of RIG-I and MDA5. We further found that TOB1-knockout led to more accumulation of mRNA transcripts of transcription factor CEBPA, and thus its protein, which further enhanced transcription of RIG-I and MDA5 genes. In addition, TOB1-knockout was shown to inhibit FMDV adsorption and internalization mediated by EGFR/ERBB2 pathway. Finally, the FMDV lethal challenge on TOB1-knockout mice confirmed that the deletion of TOB1 inhibited FMDV infection in vivo. These results identify TOB1 as a key host factor involved in FMDV infection in pigs.

The antiviral response triggered by the cGAS/STING pathway is subverted by the foot-and-mouth disease virus proteases

Propagation of viruses requires interaction with host factors in infected cells and repression of innate immune responses triggered by the host viral sensors. Cytosolic DNA sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) is a major component of the antiviral response to DNA viruses, also known to play a relevant role in response to infection by RNA viruses, including foot-and-mouth disease virus (FMDV). Here, we provide supporting evidence of cGAS degradation in swine cells during FMDV infection and show that the two virally encoded proteases, Leader (Lpro) and 3Cpro, target cGAS for cleavage to dampen the cGAS/STING-dependent antiviral response. The specific target sequence sites on swine cGAS were identified as Q140/T141 for the FMDV 3Cpro and the KVKNNLKRQ motif at residues 322–330 for Lpro. Treatment of swine cells with inhibitors of the cGAS/STING pathway or depletion of cGAS promoted viral infection, while overexpression of a mutant cGAS defective for cGAMP synthesis, unlike wild type cGAS, failed to reduce FMDV replication. Our findings reveal a new mechanism of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, based on the redundant degradation of cGAS through the concomitant proteolytic activities of two proteases encoded by an RNA virus, further proving the key role of cGAS in restricting FMDV infection.

Foot-and-Mouth Disease Virus Capsid Protein VP1 Antagonizes Type I Interferon Signaling via Degradation of Histone Deacetylase 5.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease of cloven-hoofed animals that hampers trade and production. To ensure effective infection, the foot-and-mouth disease virus (FMDV) evades host antiviral pathways in different ways. Although the effect of histone deacetylase 5 (HDAC5) on the innate immune response has previously been documented, the precise molecular mechanism underlying HDAC5-mediated FMDV infection is not yet clearly understood. In this study, we found that silencing or knockout of HDAC5 promoted FMDV replication, whereas HDAC5 overexpression significantly inhibited FMDV propagation. IFN-β and IFN-stimulated response element (ISRE) activity was strongly activated through the overexpression of HDAC5. The silencing and knockout of HDAC5 led to an increase in viral replication, which was evident by decreased IFN-β, ISG15, and ISG56 production, as well as a noticeable reduction in IRF3 phosphorylation. Moreover, the results showed that the FMDV capsid protein VP1 targets HDAC5 and facilitates its degradation via the proteasomal pathway. In conclusion, this study highlights that HDAC5 acts as a positive modulator of IFN-β production during viral infection, while FMDV capsid protein VP1 antagonizes the HDAC5-mediated antiviral immune response by degrading HDAC5 to facilitate viral replication.

Calcium Chloride Treatment Enhances Antigen Production in Foot-and-Mouth Disease Vaccines for Serotypes SAT1 and SAT3.

Foot-and-mouth disease (FMD) is a highly contagious viral infection causing acute and severe vesicular lesions in cattle and pigs, which has prompted global vaccination policies. This study presents a technique for enhancing antigen yield in SAT1 BOT and SAT3 ZIM by treatment with calcium chloride (CaCl2). We tested changes in cell viability in BHK-21 suspension cells treated with varying concentrations of CaCl2. The optimal CaCl2 concentration was determined based on antigen yield. The timing of CaCl2 supplementation relative to FMD virus inoculation was tested. Finally, the optimal medium for antigen production was identified. We observed a concentration-dependent decrease in BHK-21 cell viability at >7.5 mM CaCl2. A CaCl2 concentration of 3 mM yielded the most antigens. CaCl2 supplementation relative to FMD virus infection was optimal 2 h before or with viral inoculation. CD-BHK 21 medium supplemented with CaCl2 was the most productive medium. Specifically, SAT1 BOT and SAT3 ZIM showed improved antigen production in CD-BHK 21 medium with 3 mM CaCl2, while Provero-1 and Cellvento BHK-200 media showed no significant enhancement. Overall, CaCl2 supplementation enhanced FMD antigen productivity. This study provides a useful framework for enhancing antigen production efficiently in the FMD vaccine industry.

Circulation of foot-and-mouth disease serotypes, risk factors, and their effect on hematological and biochemical profiles among cattle and buffalo in Quetta, Balochistan, Pakistan.

Foot-and-mouth disease (FMD) is an infectious disease of cloven-hoofed animals, including buffalo, cattle, sheep, goats, and pigs, causing major economic losses to the local farmers and, overall, to the national economy of the country. This study aimed to detect FMDV serotypes in year-round FMD outbreaks, hematological and biochemical changes, and oxidative stress in FMDV-infected cattle and buffaloes in the district of Quetta, Balochistan, Pakistan, and the socioeconomic impact of FMD outbreaks on farmers. We conducted a cross-sectional study in the district of Quetta, Balochistan, Pakistan, where FMD virus (FMDV) serotypes were detected by enzyme-linked immunosorbent assay (ELISA). Hematological, biochemical, and oxidative analyses were performed by analyzing the blood of FMDV-infected and non-infected animals. Information on the associated risk factors was obtained through a structured questionnaire by interviewing farmers in each FMD-affected farm. Thirty-four out of 38 farms (89%, 95% confidence interval [CI]: 75%-97%) were positive for FMD by ELISA. Higher FMD infection was detected in farms with a herd size of <50 animals (50%, 17/34), followed by >100 animals (32%, 11/34) and 51-100 animals (18%, 6/34). Fifty-seven percent (114/200, 95% CI: 50%-64%) of animals were positive for FMD. Of these, 61% (69/114) were cattle and 39% (45/114) were buffalo. FMD positivity was higher in females (86%, 98/114) than in males (14%, 16/114) and higher in animals older than 2 years of age (52%, 59/114). On average, farmers lose U.S. dollars 3000 annually due to FMD outbreaks. Animals infected with FMDV had significantly (p ≤ 0.05) white blood cell counts and significantly (p ≤ 0.05) lower hemoglobin and total protein concentrations in buffalo and cattle, whereas infected cattle showed significantly (p ≤ 0.05) lower albumin levels. Globulin levels were lower in buffaloes infected. Alanine aminotransferase levels were lower in infected cattle (p ≤ 0.05). Creatinine levels were higher in infected buffalo (p ≤ 0.05). Urea and phosphorus concentrations were higher in FMDV-infected cattle and buffalo (p ≤ 0.05). Calcium levels were lower in infected cattle and buffalo (p ≤ 0.05). Catalase enzyme activity in infected cattle and buffaloes was significantly lower (p < 0.05). Lipid peroxidation was significantly higher in FMDV-infected cattle and buffalo (p ≤ 0.05). This study confirmed serotype O circulation among cattle and buffalo in year-long FMD outbreaks in the Quetta District of Balochistan. Blood analysis identified a parameter deviated from the normal level due to FMDV infection. In addition, the outbreak of FMD has a significant negative economic impact on livestock farmers.

Beyond the Magic of Moringa oleifera: Its Potential to Control Indonesian Serotype of Footand-Mouth-Disease Virus Replication through Inhibition of 3-Cysteine Protease

Foot-and-Mouth Disease (FMD) poses a significant threat to livestock worldwide, necessitating innovative approaches to combat its causative agent, the FMD virus (FMDV). On the other hand, Moringa oleifera is a feed alternative for cattles with numerous bioactive compounds. This paper delves into the captivating realm of Moringa oleifera (MO) bioactives and their potential in thwarting FMDV replication by targeting the essential enzyme, 3C Protease (3CP). To elucidate the inhibitory potential of these bioactives, a rigorous investigation involving molecular docking and molecular dynamics simulations was conducted. Specifically, the 3CP was modeled based on the amino acid sequence of FMDV Indonesian Serotype. Results showed that most of the compounds from MO outperformed Ribavirin as the standard therapy for FMD. Among them, Baicalin, Chlorogenic Acid, and Rutin have binding affinity -9.1, -8.1, and -8.1 kcal/mol, respectively. Those compounds also formed more hydrogen bonds than Ribavirin through their binding sites. Molecular dynamics simulation also revealed that interaction of 3CP with those compounds had minor influence on its structural stability. The conformation of those compounds is also more stable than Ribavirin, supported by more hydrogen bonds. In summary, this research highlighted the potential mechanism of MO bioactives in preventing severe FMDV infection through inhibition of viral replication.

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Experimental infection of foot and mouth disease virus (FMDV) upregulates the expression of Coxsackie and adenovirus receptor (CAR) in the myocardium of suckling mice

The relative overexpression of Coxsackie and adenoviral receptor (CAR) predisposes children to viral myocarditis. As the foot and mouth disease virus (FMDV) causes fatal myocarditis in calves, lambs, and piglets and belongs to the same family as the Coxsackie virus, we investigated the role of CAR in FMDV induced myocarditis in the suckling mice model. Swiss albino suckling mice of 5 days (n = 24) were divided into two equal groups. One group was inoculated with suckling mice adapted FMDV serotype O at 10 LD50, while the other group served as uninfected control. In addition, adult mice (n = 12) served as the control for age related CAR expression and lack of pathogenicity to FMDV. The establishment of myocarditis was confirmed by histopathological changes typical of myocarditis along with immunolocalization of FMDV antigens in the heart of suckling mice. The FMDV inoculated suckling mice group showed a significant upregulation of CAR transcripts by 2.5 folds, overexpression of CAR protein by densitometric analysis of immunoblots, and intense immunolocalization of CAR in the sarcolemma and intercalated discs of cardiomyocytes as compared to the uninfected suckling mice group and adult mice. It was concluded that FMDV infection induced overexpression of CAR in the myocardium of suckling mice.

Development of a primary cell model derived from porcine dorsal soft palate for foot-and-mouth disease virus research and diagnosis.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals that has a significant socio-economic impact. One concern associated with this disease is the ability of its etiological agent, the FMD virus (FMDV), to persist in its hosts through underlying mechanisms that remain to be elucidated. While persistence has been described in cattle and small ruminants, it is unlikely to occur in pigs. One of the factors limiting the progress in understanding FMDV persistence and, in particular, differential persistence is the lack of suitable in vitro models. A primary bovine cell model derived from the dorsal soft palate, which is the primary site of replication and persistence of FMDV in cattle, has been developed, and it seemed relevant to develop a similar porcine model. Cells from two sites of FMDV replication in pigs, namely, the dorsal soft palate and the oropharyngeal tonsils, were isolated and cultured. The epithelial character of the cells from the dorsal soft palate was then assessed by immunofluorescence. The FMDV-sensitivity of these cells was assessed after monolayer infection with FMDV O/FRA/1/2001 Clone 2.2. These cells were also grown in multilayers at the air-liquid interface to mimic a stratified epithelium susceptible to FMDV infection. Consistent with what has been shown in vivo in pigs, our study showed no evidence of persistence of FMDV in either the monolayer or multilayer model, with no infectious virus detected 28 days after infection. The development of such a model opens up new possibilities for the study and diagnosis of FMDV in porcine cells.

Antiviral mechanisms of sorafenib against foot-and-mouth disease virus via c-RAF and AKT/PI3K pathways.

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that poses a significant threat to the global livestock industry. However, specific antiviral treatments against FMDV are currently unavailable. This study aimed to evaluate the antiviral activity of anticancer drugs, including kinase and non-kinase inhibitors against FMDV replication in BHK-21 cells. Sorafenib, a multi-kinase inhibitor, demonstrated a significant dose-dependent reduction in FMDV replication. It exhibited a half maximal effective concentration (EC50) value of 2.46µM at the pre-viral entry stage and 2.03µM at the post-viral entry stage. Further intracellular assays revealed that sorafenib effectively decreased 3Dpol activity with a half maximal inhibitory concentration (IC50) of 155nM, while not affecting 3Cpro function. The study indicates that sorafenib influences host protein pathways during FMDV infection, primarily by potentiating the c-RAF canonical pathway and AKT/PI3K pathway. Molecular docking analysis demonstrated specific binding of sorafenib to the active site of FMDV 3Dpol, interacting with crucial catalytic residues, including D245, D338, S298, and N307. Additionally, sorafenib exhibited significant binding affinity to the active site motifs of cellular kinases, namely c-RAF, AKT, and PI3K, which play critical roles in the viral life cycle. The findings suggest that sorafenib holds promise as a therapeutic agent against FMDV infection. Its mechanism of action may involve inhibiting FMDV replication by reducing 3Dpol activity and regulating cellular kinases. This study provides insights for the development of novel therapeutic strategies to combat FMDV infections.

Foot-and-Mouth Disease Virus Induces Porcine Gasdermin E-Mediated Pyroptosis through the Protease Activity of 3Cpro.

Foot-and-mouth disease (FMD) is an acute, highly contagious disease of cloven-hoofed animals caused by FMD virus (FMDV). Currently, the molecular pathogenesis of FMDV infection remains poorly understood. Here, we demonstrated that FMDV infection induced gasdermin E (GSDME)-mediated pyroptosis independent of caspase-3 activity. Further studies showed that FMDV 3Cpro cleaved porcine GSDME (pGSDME) at the Q271-G272 junction adjacent to the cleavage site (D268-A269) of porcine caspase-3 (pCASP3). The inhibition of enzyme activity of 3Cpro failed to cleave pGSDME and induce pyroptosis. Furthermore, overexpression of pCASP3 or 3Cpro-mediated cleavage fragment pGSDME-NT was sufficient to induce pyroptosis. Moreover, the knockdown of GSDME attenuated the pyroptosis caused by FMDV infection. Our study reveals a novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the pathogenesis of FMDV and the design of antiviral drugs. IMPORTANCE Although FMDV is an important virulent infectious disease virus, few reports have addressed its relationship with pyroptosis or pyroptosis factors, and most studies focus on the immune escape mechanism of FMDV. GSDME (DFNA5) was initially identified as being associated with deafness disorders. Accumulating evidence indicates that GSDME is a key executioner for pyroptosis. Here, we first demonstrate that pGSDME is a novel cleavage substrate of FMDV 3Cpro and can induce pyroptosis. Thus, this study reveals a previously unrecognized novel mechanism of pyroptosis induced by FMDV infection and might provide new insights into the design of anti-FMDV therapies and the mechanisms of pyroptosis induced by other picornavirus infections.

Household production and consumption impacts of foot and mouth disease at the Uganda-Tanzania border.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that is endemic in East Africa. FMD virus infection incurs significant control costs and reduces animal productivity through weight loss, lowered milk yield, and potentially death but how household's respond to these losses may differentially affect household income and food consumption. To address this, we use unique data from a FMD outbreak to assess how household production and consumption activities change from before to during the outbreak. Data came from a 2018 survey of 254 households in selected Tanzanian wards and sub-counties in Uganda. The data includes household recall of before and during an outbreak in the past year on livestock and livestock product sales, milk and beef consumption, as well as related changes in market prices. We apply both difference-in-difference and change in difference ordinary least squares regressions with fixed effects to evaluate the impact of FMD on household production and consumption. We find that households reported the largest reductions in livestock and livestock product sales, followed by reduced milk consumption and animal market prices. The changes in household income from livestock sales appears to be driven by FMD virus infection within the household herd while changes in market prices of substitute protein sources are primary associated with changes in milk and beef consumption. The role of widespread market price effects across both infected and uninfected herds and countries, tends to suggest that stabilizing prices will likely have a large impact on household nutritional security and income generation. We also propose that promoting diversity in market activity may mitigate differing impacts on households in FMD endemic regions.

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-β, 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.

The Host Protein CAD Regulates the Replication of FMDV through the Function of Pyrimidines' De Novo Synthesis.

Foot-and-mouth disease virus (FMDV) is a single-stranded picornavirus that causes economically devastating disease in even-hooved animals. There has been little research on the function of host cells during FMDV infection. We aimed to shed light on key host factors associated with FMDV replication during acute infection. We found that HDAC1 overexpression in host cells induced upregulation of FMDV RNA and protein levels. Activation of the AKT-mammalian target of rapamycin (mTOR) signaling pathway using bpV(HOpic) or SC79 also promoted FMDV replication. Furthermore, short hairpin RNA (shRNA)-induced suppression of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), a transcription factor downstream of the AKT-mTOR signaling pathway, resulted in downregulation of FMDV RNA and protein levels. Coimmunoprecipitation assays showed that the ACTase domain of CAD could interact with the FMDV 2C protein, suggesting that the ACTase domain of CAD may be critical in FMDV replication. CAD proteins participate in de novo pyrimidine synthesis. Inhibition of FMDV replication by deletion of the ACTase domain of CAD in host cells could be reversed by supplementation with uracil. These results revealed that the contribution of the CAD ACTase domain to FMDV replication is dependent on de novo pyrimidine synthesis. Our research shows that HDAC1 promotes FMDV replication by regulating de novo pyrimidine synthesis from CAD via the AKT-mTOR signaling pathway. IMPORTANCE Foot-and-mouth disease virus is an animal virus of the Picornaviridae family that seriously harms the development of animal husbandry and foreign trade of related products, and there is still a lack of effective means to control its harm. Replication complexes would generate during FMDV replication to ensure efficient replication cycles. 2C is a common viral protein in the replication complex of Picornaviridae virus, which is thought to be an essential component of membrane rearrangement and viral replication complex formation. The host protein CAD is a key protein in the pyrimidines de novo synthesis. In our research, the interaction of CAD and FMDV 2C was demonstrated in FMDV-infected BHK-21 cells, and it colocalized with 2C in the replication complex. The inhibition of the expression of FMDV 3D protein through interference with CAD and supplementation with exogenous pyrimidines reversed this inhibition, suggesting that FMDV might recruit CAD through the 2C protein to ensure pyrimidine supply during replication. In addition, we also found that FMDV infection decreased the expression of the host protein HDAC1 and ultimately inhibited CAD activity through the AKT-mTOR signaling pathway. These results revealed a unique means of counteracting the virus in BHK-21 cells lacking the interferon (IFN) signaling pathway. In conclusion, our study provides some potential targets for the development of drugs against FMDV.

Heterogeneity and Recombination of Foot-and-Mouth Disease Virus during Multi-Strain Coinfection of Cattle.

Superinfection of cattle persistently infected with foot-and-mouth disease virus (FMDV), with a heterologous FMDV strain has been shown to generate novel recombinant viruses. In this study, we investigated the pathogenesis events within specific tissues associated with FMDV coinfections in cattle subjected to either simultaneous or serial exposure to two distinct strains of FMDV. Both strains of FMDV (one each of serotypes O and A) were similarly localized to the nasopharyngeal mucosa during the early stages of infection. However, while no recombinant FMDV genomes were recovered from simultaneously coinfected cattle, interserotypic recombinants were isolated from nasopharyngeal tissue samples obtained at 48 h after heterologous superinfection of a persistently infected FMDV carrier. Additionally, analysis of FMDV genomes obtained from replicate nasopharyngeal tissue samples demonstrated that adjacent segments of the mucosa were sometimes infected by distinct viruses, demonstrating a multifocal and heterogeneous distribution of FMDV infection during primary and persistent phases of infection. This work indicates that superinfection of FMDV carriers may be an important source of emergent recombinant strains of FMDV in areas where multiple strains are co-circulating. IMPORTANCE Foot-and-mouth disease (FMD) is a socioeconomically impactful livestock disease with a complex epidemiology and ecology. Although recombinant viruses have been identified in field samples, the mechanisms of emergence of those viruses have never been elucidated. This current study demonstrates how serial infection of cattle with two distinct serotypes of FMD virus (FMDV) leads to rapid generation of recombinant viruses in the upper respiratory tracts of infected animals. This finding is particularly relevant in relation to the management of persistently infected FMDV carrier cattle that can maintain subclinical FMDV infection for months to years after an initial infection. Such carrier animals may function as mixing vessels that facilitate the emergence of novel recombinant FMDV strains in areas where multiple virus strains are in circulation.

IL-10 Promotes CXCL13 Expression in Macrophages Following Foot-and-Mouth Disease Virus Infection.

Foot-and-mouth disease (FMD) is one of the most contagious livestock diseases in the world, posing a constant global threat to the animal trade and national economies. The chemokine C-X-C motif chemokine ligand 13 (CXCL13), a biomarker for predicting disease progression in some diseases, was recently found to be increased in sera from mice infected with FMD virus (FMDV) and to be associated with the progression and severity of the disease. However, it has not yet been determined which cells are involved in producing CXCL13 and the signaling pathways controlling CXCL13 expression in these cells. In this study, the expression of CXCL13 was found in macrophages and T cells from mice infected with FMDV, and CXCL13 was produced in bone-marrow-derived macrophages (BMDMs) by activating the nuclear factor-kappaB (NF-κB) and JAK/STAT pathways following FMDV infection. Interestingly, CXCL13 concentration was decreased in sera from interleukin-10 knock out (IL-10-/-) mice or mice blocked IL-10/IL-10R signaling in vivo after FMDV infection. Furthermore, CXCL13 was also decreased in IL-10-/- BMDMs and BMDMs treated with anti-IL-10R antibody following FMDV infection in vitro. Lastly, it was demonstrated that IL-10 regulated CXCL13 expression via JAK/STAT rather than the NF-κB pathway. In conclusion, the study demonstrated for the first time that macrophages and T cells were the cellular sources of CXCL13 in mice infected with FMDV; CXCL13 was produced in BMDMs via NF-κB and JAK/STAT pathways; and IL-10 promoted CXCL13 expression in BMDMs via the JAK/STAT pathway.

Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-β production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

Picornavirus infection enhances aspartate by the SLC38A8 transporter to promote viral replication.

Foot-and-mouth disease, a class of animal diseases, is caused by foot-and-mouth disease virus (FMDV). The metabolic changes during FMDV infection remain unclear. Here, PK-15 cells, serum, and tonsils infected with FMDV were analyzed by metabolomics. A total of 284 metabolites in cells were significantly changed after FMDV infection, and most of them belong to amino acids and nucleotides. Further studies showed that FMDV infection significantly enhanced aspartate in vitro and in vivo. The amino acid transporter solute carrier family 38 member 8 (SLC38A8) was responsible for FMDV-upregulated aspartate. Enterovirus 71 (EV71) and Seneca Valley virus (SVV) infection also enhanced aspartate by SLC38A8. Aspartate aminotransferase activity was also elevated in FMDV-, EV71-, and SVV-infected cells, which may lead to reversible transition between the TCA cycle and amino acids synthesis. Aspartate and SLC38A8 were essential for FMDV, EV71, and SVV replication in cells. In addition, aspartate and SLC38A8 also promoted FMDV and EV71 replication in mice. Detailed analysis indicated that FMDV infection promoted the transfer of mTOR to lysosome to enhance interaction between mTOR and Rheb, and activated PI3K/AKT/TSC2/Rheb/mTOR/p70S6K1 pathway to promote viral replication. The mTORC1 signaling pathway was responsible for FMDV-induced SLC38A8 protein expression. For the first time, our data identified metabolic changes during FMDV infection. These data identified a novel mechanism used by FMDV to upregulate aspartate to promote viral replication and will provide new perspectives for developing new preventive strategies.

Antiviral Effect of Manganese against Foot-and-Mouth Disease Virus Both in PK15 Cells and Mice.

Foot-and-mouth disease (FMD) is an acute contagious disease of cloven-hoofed animals such as cattle, pigs, and sheep. Current emergency FMD vaccines are of limited use for early protection because their protective effect starts 7 days after vaccination. Therefore, antiviral drugs or additives are used to rapidly stop the spread of the virus during FMD outbreaks. Manganese (Mn2+) was recently found to be an important substance necessary for the host to protect against DNA viruses. However, its antiviral effect against RNA viruses remains unknown. In this study, we found that Mn2+ has antiviral effects on the FMD virus (FMDV) both in PK15 cells and mice. The inhibitory effect of Mn2+ on FMDV involves NF-κB activation and up-regulation of interferon-stimulated genes. Animal experiments showed that Mn2+ can be highly effective in protecting C57BL/6N mice from being infected with FMDV. Overall, we suggest Mn2+ as an effective antiviral additive for controlling FMDV infection.