EV71 is highly infectious to the central nervous system, resulting in a series of characteristic clinical symptoms including encephalitis, aseptic meningitis, acute flaccid paralysis, herpangina, acute hemorrhagic conjunctivitis, myoclonic jerks, headache, fever, and vomiting, among which HFMD and herpangina are the most common. Most EV infections are generally asymptomatic (approximately 50–80%) or produce mild, cold-like symptoms, and affected patients recover naturally and produce antibodies. However, severely ill patients infected with EVs are at high risk of dying from cardiopulmonary failure and extensive brain stem injury. EV71 mostly affects the nervous system in children Table 2). 3.1. Repositioning of Antifungal DrugsGao et al., Xu et al., and Chonsaeng et al. recently considered the repositioning of antifungal drugs and showed that itraconazole (ITZ) (1), micafungin (2), and amphotericin B (3) had potential pharmacological activities against EV71 infection (Figure 1). ITZ was detected by high-throughput screening assay [59] of 1280 clinical compounds in the FDA-approved drug library to detect potentially effective drugs, among which ITZ demonstrated good pharmacokinetics and safety [59]. ITZ is a new-generation triazole and is a highly-efficient, broad-spectrum antifungal drug mainly used to treat systemic infections caused by deep fungi. It can be combined with the fungal cytochrome P450 isozyme to inhibit the synthesis of ergosterol. ITZ has also been reported to act as a broad-spectrum enterovirus inhibitor, with an IC50 of 1.15 μM. Mutation of the 3A protein can cause ITZ resistance in EV71, which strongly inhibits viral RNA replication or polyprotein processing [59].In addition, Chonsaeng et al. identified micafungin among 968 FDA-approved drugs. Micafungin is an echinocandin antifungal drug from Coleophoma empetri obtained by chemical synthesis, which shows good inhibitory activity against Candida spp., including Candida albicans, as well as in vitro inhibitory activity against Aspergillus. The researchers speculated that micafungin might target any step in early viral infection, with an EC50 of 2–10 μM. Although the mechanism of action of micafungin against EV71 infection remains unclear, it does not appear to involve internal ribosome entry site (IRES)-dependent translation or polyprotein processing involving 3Cpro, 2C, and 3A proteins [60].Amphotericin B was found to be a potentially useful drug by Xu et al. on the basis of combination therapy used to treat co-infection by fungi and viruses [61]. Amphotericin B is a polyene antifungal drug used for systemic infections or infections of internal organs caused by Cryptococcus and Aspergillus. Amphotericin B can effectively inhibit the production of EV71, with an EC50 of 1.75 μM in Rhabdomyosarcoma (RD) cells and 0.32 μM in HEK293 cells. Amphotericin B was shown to impair the binding and internalization of EV71 virus to host cells, using western blotting, quantitative real-time polymerase chain reaction, and virus-binding assay [61]. 3.2. Repositioning of Antibacterial Drugs

CVA16 belongs to the enterovirus genus of the Picornaviridae family and is one of the main pathogens of HFMD. CVA16 has recently been shown to cause secondary infections of the brain, lung, and heart and may even lead to fatal complications such as pneumonia, myocarditis, and refractory shock.

Zeng et al. discovered that the clinically marketed antibiotic azithromycin (4) could be used to treat EV71 and CVA16 infections (Figure 2). Azithromycin is mainly used to treat respiratory tract, skin soft tissue, and urogenital system infections but has also demonstrated antiviral activity against EV71. Azithromycin significantly reduced EV71 RNA and protein levels and probably acted by interfering with viral RNA replication [62]. Notably, azithromycin has been clinically proven to be safe in pregnant women, newborns, and young children and may thus be particularly useful, given that HFMD mostly occurs in children under 5 years of age. Notably, the combination of azithromycin with the macrolide antibiotic spiramycin (SPM) (5) inhibited the replication of EV71 and CVA16 (Figure 2), thereby exerting antiviral effects [62]. Their antiviral mechanisms appear to be similar, and EV71 mutant strains resistant to SPM show similar resistance to azithromycin. Although the specific mechanisms of these drugs are unknown, they are likely to produce antiviral effects by inhibiting virus RNA replication.Liao et al. found that the antibiotic minocycline (6) had anti-inflammatory and immunomodulatory properties in infectious and inflammatory neurological disease models (Figure 2). They, therefore, carried out a series of in vitro and in vivo experiments to explore its effect on EV71 infection. Minocycline is a broad-spectrum antibacterial tetracycline antibiotic that can be combined with tRNA to achieve bacteriostatic effects and has the strongest antibacterial effect among tetracycline antibiotics. Unlike milrinone (drug 19), which is mainly used to treat complications of EV71 infection with no obvious anti-viral effect [63], minocycline reduced virus replication, specifically VP0 and VP2, while double-dose treatment suppressed cytokine production and viral protein expression in EV71-infected THP-1 cells [64]. 3.3. Repositioning of Cardiovascular Drugs

CVB3 is one of the most important causative factors of viral myocarditis, accounting for 50% of cases. CVBs viruses can directly damage host cells, leading to an inflammatory host response to viral infection, resulting in target organ tissue damage and dysfunction. CVBs can harness different host cell processes including kinases, host cell-killing and cell-eating machineries, matrix metalloproteinases, and miRNAs to promote disease. Although recent improvements in treatment methods have significantly improved disease outcomes, some patients still have poor treatment responses and relapse. The incidence and mortality of CVB3-related viral myocarditis are increasing year by year. The clinical symptoms of the disease vary in severity, ranging from no obvious symptoms to shortness of breath, fatigue, chest tightness, cardiomyopathy, and even congestive heart failure and cardiogenic shock. Unfortunately, there is still no effective treatment for this disease. However, some FDA-approved drugs have been repurposed for viral myocarditis caused by CVB3 infection. These drugs have different mechanisms of action and different therapeutic effects against viral myocarditis. Since these drugs have already passed time-consuming and laborious clinical trials and have been shown to be safe, the potential repurposing of these drugs for CVB3 infection or viral myocarditis may save unnecessary costs and accelerate the research and development time.

Lovastatin (7), bosentan (8), and valsartan (9) inhibit the replication of CVB3 by down-regulating the expression of coxsackie-adenovirus receptor (CAR) mRNA and protein (Figure 3). CVB3 requires the human CAR to infect the myocardium. Lovastatin is a lipid-lowering drug, mainly used for the treatment of hypercholesterolemia, especially in patients with elevated low-density lipoprotein (type II). Bosentan is a specific and competitive low-molecular weight dual endothelin receptor blocker, mainly used for the treatment of pulmonary hypertension. Valsartan is an angiotensin II receptor antagonist that selectively blocks the binding of angiotensin II and angiotensin II type 1 receptor receptors, thereby inhibiting vasoconstriction and aldosterone release, resulting in a hypotensive effect. These three drugs, with completely different pharmacological effects, play important roles in inhibiting the replication of CVB3 through a similar mechanism. Werner et al. found that lovastatin decreased CAR mRNA and protein expression levels by up to 80% and 19% (p65], in a concentration-dependent manner. In contrast, bosentan drastically decreased CAR mRNA levels in HeLa cells and human umbilical vein endothelial cells (HUVECs) by 80.2% (±4.6%) and by 66.3% (±12.6%), respectively [66], while valsartan also reduced CAR mRNA in HUVECs and HeLa cells by up to 68.1% (±8.7%) and 51.4% (±8.8%), respectively [66].Assuming that CVB3 enters the host cell through the CAR, Gazina et al. discovered that amiloride inhibited the enzymatic activity of CVB3 3Dpol in vitro [67], affecting RNA elongation. Amiloride (10) is mainly used to treat liver cirrhosis, edema, and primary aldosteronism (Figure 3). It has strong sodium-excretion and potassium-sparing diuretic effects and is mainly used to treat mild to moderate hypertension. Amiloride can block various ion channels, including the Na+/H+ exchanger, acid-sensitive ion channels, Na+/Ca2+ exchanger, voltage-gated Na+ channels, and Ca2+ channels. Moreover, Amiloride can act as a competitive inhibitor, competing with incoming nucleoside triphosphates and Mg2+, resulting in inhibition of CVB3 RNA replication [67].β-adrenoreceptor agonists can activate the p38 mitogen-activated protein kinase (MAPK) pathway, leading to the expression of proinflammatory cytokines, further triggering inflammation and apoptosis. Carvedilol (11) is a β-adrenoreceptor antagonist that blocks β1- and β2-adrenoreceptors, inhibits activation of the p38 MAPK pathway [68], and then down-regulates the expression of proinflammatory cytokines such as interleukin (IL)-1β and IL-8 (Figure 3). Wang et al. also compared the roles of carvedilol and metoprolol for the treatment of viral myocarditis [68] and found that carvedilol improved cardiac contractility and diastolic function, while metoprolol only improved contractile function, indicating that carvedilol protects heart function by mechanisms other than inhibiting β-adrenergic receptors. It may exert a variety of pharmacological effects, such as anti-oxidative stress and myocardial remodeling, but further studies are needed to confirm these effects.Apart from drug screening, evidence-based medicine and retrospective analysis of cases may also help researchers to identify approved drugs that can be repurposed for EV71 infection, such as carvedilol, which blocks calcium channels and is suitable for the treatment of symptomatic heart failure and essential hypertension. Gong et al. found that carvedilol reduced norepinephrine and epinephrine levels, diastolic and systolic pressure, blood glucose level, heart rate, body temperature, and leukocyte count in children with EV71 HFMD [69], suggesting that carvedilol may alleviate the effects of HFMD.In contrast to carvedilol, formoterol (12) is a β2 receptor agonist that can activate the p38 MAPK pathway and which also inhibited CVB3-induced myocarditis [70] (Figure 4). Rachel et al. found that high concentrations of β2 receptor agonists and inhibitors had no antiviral effects against CVB3 infection, indicating that formoterol did not exert its anti-CVB3 effect by stimulating the β2 receptor. Although the antiviral mechanism of formoterol remains unclear, it acts as a pan-enterovirus inhibitor.In addition to drugs that act on β-receptors, nicotine (13) may also improve inflammation (Figure 4). Nicotine is an alkaloid found in Solanaceae plants (Solanum) and an α7-nicotinic acetylcholine receptor antagonist, which mainly acts on α-cholinergic receptors. Zhao et al. used nicotine in a mouse model of CVB3 infection and found that the survival rate of BALB/C mice in the nicotine group after 14 days was 80%, compared with only 45% in the drug-free group [71]. Nicotine also significantly reduced myocardial damage and cell infiltration and down-regulated proinflammatory cytokines such as IL-6 and IL-17A. These findings suggest that nicotine can effectively improve myocarditis caused by CVB3 [71].There are many types of proinflammatory cytokines, and the generation of nitric oxide (NO) is likely to cause myocardial necrosis and contractile dysfunction, suggesting that suppression of NO may reduce the severity of myocarditis. Ivabradine (14) and olmesartan (15) inhibited NO synthesis by inhibiting inducible NO synthase, thereby treating viral myocarditis (Figure 4). Ivabradine (trade name: Corlanor), a selective atrionector-specific If current blocker that slows sinus rhythm, was approved by the FDA in 2015 for the treatment of patients with heart failure through a priority review process. Ivabradine also reduces myocardial damage and down-regulates the expression of proinflammatory cytokines [72]. Olmesartan is an angiotensin II receptor antagonist that can be used as an antihypertensive drug.The FDA-approved drugs atorvastatin (16) and losartan (17) can also be used as immunomodulators to treat viral myocarditis (Figure 4). Atorvastatin reduced the abnormal expression of tumor necrosis factor-a and IFN-c and restored the expression of connexins such as Cx43 and Cx45 [73], thereby reducing the rate of myocardial necrosis and effectively treating viral myocarditis. Atorvastatin (trade name: Lipitor) is a lipid-lowering drug developed by Pfizer, which is mainly used to treat hypercholesterolemia and coronary heart disease. Losartan can down-regulate the expression of Th17 cells and stimulate Th1 cells to secrete relevant cytokines. Losartan significantly reduced mortality in cytomegalovirus (CMV)-infected mice from 40.0% to 12.5% [74]. Losartan (trade name: Cozaar), as the first angiotensin II receptor antagonist class antihypertensive drug, blocks the key sex hormone angiotensin II to regulate blood pressure.Captopril (18) is an angiotensin-converting enzyme inhibitor used to treat hypertension and certain types of congestive heart failure (Figure 4). It can reduce myocardial calcification and fibrosis by an unknown mechanism, possibly related to resistance to mitochondrial damage [75]. Milrinone (19) has been used to treat pulmonary edema, as a potentially fatal complication of EV71 infection (Figure 4). Milrinone is an FDA-approved drug that enhances myocardial contractility and directly expands blood vessels. It is used clinically for the treatment of chronic congestive heart failure and intractable heart failure. Although it cannot directly inhibit the production or replication of EV71, milrinone exerts immunomodulatory and anti-inflammatory effects in the management of systemic inflammatory response to severe EV71 infection and has demonstrated good clinical therapeutic efficacy against EV71-related brainstem encephalitis [63]. 3.7. Repositioning of Other DrugsZeng et al. also found that the ability of azithromycin (4) to inhibit EV71 infection was comparable to that of chloroquine (27). Chloroquine has been used clinically since 1944, initially to treat malaria, but its use has gradually expanded, and chloroquine was used to treat rheumatoid arthritis in 1951. Shang et al. reported that 1.2 μM of chloroquine resulted in a 104-fold reduction in EV71 RNA synthesis [83], while Shih et al. [84] showed that it blocked the uncoating of EV71 and reduced viral RNA synthesis by >90%. These findings suggest that chloroquine may be an effective EV71 virus inhibitor (Figure 8).Methylene blue (28) is an aromatic heterocyclic compound that is used as a chemical indicator, dye, biological stain, and drug. It has been shown to destroy EV71 viral proteins and genome in a photodynamically inactive and dose-dependent manner [85] (Figure 8).Arsenic trioxide (29) was shown by Ylva et al. to reduce the viral load of CVB3 RNA in the pancreas through an unknown mechanism [86] (Figure 8). Arsenic trioxide is an odorless and tasteless creamy white powder and one of the oldest known poisons. It is highly toxic, and close attention should thus be paid to the dosage to prevent unnecessary side effects.Cyclosporin A (30) is mainly used to prevent rejection of liver, kidney, and heart grafts (Figure 8). It inhibits the opening of mitochondrial permeability transition pores by inhibiting cyclophilin D [87]. ABCC6 mutation is associated with myocardial calcification by increasing the susceptibility of the mitochondria to calcium, etc., resulting in calcification. Cyclosporin A can thus reduce the degree of myocardial calcification by inhibiting mitochondrial permeability transition pores.In addition, cholic acid (31) 10 μg/mL was shown to reduce production of the viral capsid protein VP1 and inhibit cleavage of the translation initiation factor eIF4G1 [88] (Figure 8). Cholic acid (trade name: Cholbam) is present in bile in cattle, sheep, and pigs and has been approved by the FDA for the treatment of bile acid synthesis disorders and peroxisomal disorders. Moreover, cholic acid inhibited extracellular signal-regulated kinase (ERK) signaling in CVB3-infected HeLa cells [88]. CVB3 infection can cause persistent activation of the host ERK pathway, and inhibition of this pathway using ERK pathway-specific inhibitors, such as PD098059, can significantly reduce the production of related progeny viruses. Cholic acid plays a similar role to PD098059.Ren et al. selected suramin (32) from 1102 approved drugs. Suramin is used to treat sleeping sickness caused by trypanosomes and has also demonstrated good inhibitory effects against EV71 and CVA16 [89] (Figure 8). Suramin showed no cytotoxicity at concentrations >1 mM, and its original role as a pediatric drug prioritized the possibility of it being repositioned as a treatment for HFMD. Further studies found that structural analogs of suramin, including sulfonated and sulfated compounds, also inhibited the proliferation of EV71, suggesting promising potential and indicating that in addition to the repositioned drug, its structural analogs should also be investigated to identify more effective drug structures. Wang et al. [90] further studied the possible anti-EV71 mechanism of suramin and showed that it inhibited the proliferation of EV71 in both early and late stages of its life cycle and prevented EV71 from attaching to host cells, thereby affecting its entry.