Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome.

Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21 de novo. The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses. IMPORTANCE As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.

3,4-Dicaffeoylquinic Acid from the Medicinal Plant Ilex kaushue Disrupts the Interaction Between the Five-Fold Axis of Enterovirus A-71 and the Heparan Sulfate Receptor.

While infections by enterovirus A71 (EV-A71) are generally self-limiting, they can occasionally lead to serious neurological complications and death. No licensed therapies against EV-A71 currently exist. Using anti-virus-induced cytopathic effect assays, 3,4-dicaffeoylquinic acid (3,4-DCQA) from Ilex kaushue extracts was found to exert significant anti-EV-A71 activity, with a broad inhibitory spectrum against different EV-A71 genotypes. Time-of-drug-addition assays revealed that 3,4-DCQA affects the initial phase (entry step) of EV-A71 infection by directly targeting viral particles and disrupting viral attachment to host cells. Using resistant virus selection experiments, we found that 3,4-DCQA targets the glutamic acid residue at position 98 (E98) and the proline residue at position 246 (P246) in the 5-fold axis located within the VP1 structural protein. Recombinant viruses harboring the two mutations were resistant to 3,4-DCQA-elicited inhibition of virus attachment and penetration into human rhabdomyosarcoma (RD) cells. Finally, we showed that 3,4-DCQA specifically inhibited the attachment of EV-A71 to the host receptor heparan sulfate (HS), but not to the scavenger receptor class B member 2 (SCARB2) and P-selectin glycoprotein ligand-1 (PSGL1). Molecular docking analysis confirmed that 3,4-DCQA targets the 5-fold axis to form a stable structure with the E98 and P246 residues through noncovalent and van der Waals interactions. The targeting of E98 and P246 by 3,4-DCQA was found to be specific; accordingly, HS binding of viruses carrying the K242A or K244A mutations in the 5-fold axis was successfully inhibited by 3,4-DCQA.The clinical utility of 3,4-DCQA in the prevention or treatment of EV-A71 infections warrants further scrutiny. IMPORTANCE The canyon region and the 5-fold axis of the EV-A71 viral particle located within the VP1 protein mediate the interaction of the virus with host surface receptors. The three most extensively investigated cellular receptors for EV-A71 include SCARB2, PSGL1, and cell surface heparan sulfate. In the current study, a RD cell-based anti-cytopathic effect assay was used to investigate the potential broad spectrum inhibitory activity of 3,4-DCQA against different EV-A71 strains. Mechanistically, we demonstrate that 3,4-DCQA disrupts the interaction between the 5-fold axis of EV-A71 and its heparan sulfate receptor; however, no effect was seen on the SCARB2 or PSGL1 receptors. Taken together, our findings show that this natural product may pave the way to novel anti-EV-A71 therapeutic strategies.

Artemisia capillaris inhibited enterovirus 71-induced cell injury by preventing viral internalization.

Artemisia capillaris (A. capillaris) is a common herbal drug used for thousands years in ancient China. A. capillaris has been empirically used to manage hand-foot-mouth disease (HFMD), which is commonly caused by enterovirus 71 (EV71). EV71 can cause meningoencephalitis with mortality and neurologic sequelae without effective management. It is presently unknown whether A. capillaris is effective against EV71 infection. To test the hypothesis that it could protect cells from EV71-induced injury, a hot water extract of A. capillaris was tested in human foreskin fibroblast cells (CCFS-1/KMC) and human rhabdomyosarcoma cells (RD cells) by plaque reduction assay and flow cytometry. Inhibition of viral replication was examined by reverse quantitative RT-PCR (qRT-PCR). Its effect on translations of viral proteins (VP0, VP1, VP2, protease 2B and 3AB), and apoptotic proteins were examined by western blot. A. capillaris was dose-dependently effective against EV71 infection in both CCFS-1/KMC cells and RD cells by inhibiting viral internalization. However, A. capillaris was minimally effective on viral attachment, VP2 translation, and inhibition of virus-induced apoptosis. Further isolation of effective molecules is needed. In conclusion, A. capillaris has anti-EV71 activity mainly by inhibiting viral internalization. A. capillaris would be better to manage EV71 infection in combination with other agents.

Inhibition of EV71 by curcumin in intestinal epithelial cells.

EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.

Flos Farfarae Inhibits Enterovirus 71-Induced Cell Injury by Preventing Viral Replication and Structural Protein Expression.

Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.

Antiviral activity of Paulownia tomentosa against enterovirus 71 of hand, foot, and mouth disease.

The bark, leaves, and flowers of Paulownia trees have been used in traditional Chinese medicine to treat infectious and inflammatory diseases. We investigated the antiviral effects of Paulownia tomentosa flowers, an herbal medicine used in some provinces of P. R. China for the treatment of skin rashes and blisters. Dried flowers of P. tomentosa were extracted with methanol and tested for antiviral activity against enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), the predominant etiologic agents of hand, foot, and mouth disease in P. R. China. The extract inhibited EV71 infection, although no effect was detected against CAV16 infection. Bioactivity-guided fractionation was performed to identify apigenin as an active component of the flowers. The EC50 value for apigenin to block EV71 infection was 11.0 µM, with a selectivity index of approximately 9.3. Although it is a common dietary flavonoid, only apigenin, and not similar compounds like naringenin and quercetin, were active against EV71 infection. As an RNA virus, the genome of EV71 has an internal ribosome entry site that interacts with heterogeneous nuclear ribonucleoproteins (hnRNPs) and regulates viral translation. Cross-linking followed by immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that EV71 RNA was associated with hnRNPs A1 and A2. Apigenin treatment disrupted this association, indicating that apigenin suppressed EV71 replication through a novel mechanism by targeting the trans-acting factors. This study therefore validates the effects of Paulownia against EV71 infection. It also yielded mechanistic insights on apigenin as an active compound for the antiviral activity of P. tomentosa against EV71 infection.

Rupintrivir is a promising candidate for treating severe cases of enterovirus-71 infection: evaluation of antiviral efficacy in a murine infection model.

Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.

Development and characterization of a stable eGFP enterovirus 71 for antiviral screening.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5' untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.