The nucleoside analog 4'-fluorouridine suppresses the replication of multiple enteroviruses by targeting 3D polymerase.

Human enteroviruses are the major pathogens causing hand-foot-and-mouth disease in infants and young children throughout the world, and infection with enterovirus is also associated with severe complications, such as aseptic meningitis and myocarditis. However, there are no antiviral drugs available to treat enteroviruses infection at present. In this study, we found that 4'-fluorouridine (4'-FlU), a nucleoside analog with low cytotoxicity, exhibited broad-spectrum activity against infections of multiple enteroviruses with EC50 values at low micromolar levels, including coxsackievirus A10 (CV-A10), CV-A16, CV-A6, CV-A7, CV-B3, enterovirus A71 (EV-A71), EV-A89, EV-D68, and echovirus 6. With further investigation, the results indicated that 4'-FlU directly interacted with the RNA-dependent RNA polymerase of enterovirus, the 3D pol, and impaired the polymerase activity of 3D pol, hence inhibiting viral RNA synthesis and significantly suppressing viral replication. Our findings suggest that 4'-FlU could be promisingly developed as a broad-spectrum direct-acting antiviral agent for anti-enteroviruses therapy.

Proteomic and metabonomic analysis uncovering Enterovirus A71 reprogramming host cell metabolic pathway.

Enterovirus A71 (EV71) infection can cause hand, foot, and mouth disease (HFMD) and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in rhabdomyosarcoma (RD) cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 upregulated proteins and 12 downregulated proteins. The upregulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Alpha-enolase (ENO1) protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. The glycolysis pathway was inhibited by knocking down ENO1 or the use of a glycolysis inhibitor (dichloroacetic acid [DCA]); and we found that EV71 infection was inhibited by depleting ENO1 or using DCA. Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 may be a potential target against EV71, and DCA could act as an inhibitor of EV71.

A disulfiram-loaded electrospun poly(vinylidene fluoride) nanofibrous scaffold for cancer treatment.

Disulfiram (DSF), an FDA approved drug for the treatment of alcoholism, has shown its effectiveness against diverse cancer types. Thus, we developed a disulfiram-loaded scaffold using the electrospinning method to enhance the stability of DSF and to facilitate its appropriate distribution to tumor tissues. The drug release profile of the disulfiram-loaded scaffold was examined by high-performance liquid chromatography. We obtained mechanical and morphological characterizations of A549 cells treated with different scaffolds by various techniques to evaluate its antitumor properties. This work revealed that the cells after the treatment with the disulfiram-loaded scaffold exhibited a lower height and a larger elastic modulus compared with the untreated cells and those treated with the neat electrospun fibers. The changes were the indicators of cell apoptosis. Taken collectively, the results indicate that DSF was successfully incorporated into the electrospun fibers, and the disulfiram-loaded scaffold has great potential for inhibiting the regional recurrence of cancer.

Enhanced human enterovirus 71 infection by endocytosis inhibitors reveals multiple entry pathways by enterovirus causing hand-foot-and-mouth diseases.

BACKGROUND: Human enterovirus 71 (EV71) was previously known to enter cells through clathrin or caveolar mediated endocytic pathways. However, we observed chlorpromazine (CPZ) or dynasore (DNS), which inhibit clathrin and dynamin mediated endocytosis, did not suppress EV71 cell entry in particular cell types. So the current knowledge of entry mechanisms by EV71 is not complete. METHODS: Viral infection was examined by flow cytometry or end-point dilution assays. Viral entry was monitored by immunofluorescence or pseudoviral infections. Various inhibitors were utilized for manipulating endocytic pathways. Cellular proteins were knockdown by siRNA. RESULTS: CPZ and DNS did not inhibit but rather enhance viral infection in A549 cells, while they inhibited infections in other cells tested. We further found CPZ did not affect EV71 binding to target cells and failed to affect viral translation and replication, but enhanced viral entry in A549 cells. Immunofluorescence microscopy further confirmed this increased entry. Using siRNA experiment, we found that the enhancement of EV71 infection by CPZ did not require the components of clathrin mediated endocytosis. Finally, CPZ also enhanced infection by Coxackivirus A16 in A549 cells. CONCLUSIONS: CPZ and DNS, previously reported as EV71 entry inhibitors, may rather lead to increased viral infection in particular cell types. CPZ and DNS increased viral entry and not other steps of viral life cycles. Therefore, our study indicated an unknown dynamin-independent entry pathway utilized by enteroviruses that cause Hand-Foot-and-Mouth Diseases.

A cysteine protease inhibitor GC376 displays potent antiviral activity against coxsackievirus infection.

Infection with coxsackievirus A10 (CV-A10) can cause hand-foot-mouth disease and is also associated with severe complications, including viral pneumonia, aseptic and viral meningitis. Coxsackievirus infection may also play a role in the pathogenesis of acute myocardial infarction and in the increased risk of type 1 diabetes mellitus in adults. However, there are no approved vaccines or direct antiviral agents available to prevention or treatment of coxsackievirus infection. Here, we reported that GC376 potently inhibited CV-A10 infection in different cell lines without cytotoxicity, significantly suppressed production of viral proteins, and strongly reduced the yields of infectious progeny virions. Further study indicated that GC376, as viral 3C protease inhibitor, had the potential to restrain the cleavage of the viral polyprotein into individually functional proteins, thus suppressed the replication of CV-A10. Furthermore, the drug exhibited antiviral activity against coxsackieviruses of various serotypes including CV-A6, CV-A7 and CV-A16, suggesting that GC376 is a broad-spectrum anti-coxsackievirus inhibitor and the 3C protease is a promising target for developing anti-coxsackievirus agents.

Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade.

Enterovirus A (EV-A) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc., revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A. In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity.

The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene.

Coxsackievirus A16 (CA16) belongs to the Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.

Preparation and verification of a monoclonal antibody against a conserved linear epitope in enterovirus A protein 2C.

Enterovirus A (EV-A) species are the main agents responsible for hand, foot, and mouth disease (HFMD), a serious public health concern. Lack of appropriate reagents prevents the mechanistic study of these virus infections. 2C protein, a non-structural protein of Enterovirus, is crucial for viral replication and antiviral immunity. Here, preparation and testing of a monoclonal antibody by immunizing mice with Coxsackievirus A10 protein 2C (CVA10-2C) was reported. This antibody could identify most EV-A types, both conventional and unconventional groups. We also mapped the antibody epitope SLATGIIARA, which is highly conserved in EV-A species and located in the ATPase domain. Some key amino acids include G140, I141, I142, and R144. In conclusion, we generated a recombinant monoclonal antibody against multiple EVA types and confirmed its performance, which may facilitate the future study of Enterovirus A infection and many potential applications, such as the diagnosis of pathogen or the development of antiviral therapies.

The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10.

Coxsackievirus A10 (CVA10) is one of the major etiological agents of hand, foot, and mouth disease. There are no vaccine and antiviral drugs for controlling CVA10 infection. Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals. Here, two infectious clones for the prototype and a Myc-tagged CVA10 were constructed. Viable CVA10 viruses were harvested by transfecting the viral mRNA into human rhabdomyosarcoma (RD) cells. Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally. We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter, respectively. Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293T (HEK293T) cells led to the production of single round infectious particles (SRIPs). Based on CVA10 replicon RNA, SRIPs with either the enterovirus A71 (EVA71) capsid or the CVA10 capsid were generated. Infection by EVA71 SRIPs required SCARB2, while CVA10 SRIPs did not. Finally, we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme (HHRib) before the 5'-untranslated region (UTR). In summary, reverse genetic tools for prototype strain of CVA10, including both the infectious clone and the SRIPs system, were successfully established. These tools will facilitate the basic and translational study of CVA10.