key: cord-0998261-cijj983j
authors: Zhong, Ting; Zhang, Li-ying; Wang, Zeng-yan; Wang, Yue; Song, Feng-mei; Zhang, Ya-hong; Yu, Jing-hua
title: Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment
date: 2016-11-14
journal: Acta Pharmacologica Sinica
DOI: 10.1038/aps.2016.110
sha: 03906ffef2d37362b598ee88ce3f13e7aebe6011
doc_id: 998261
cord_uid: cijj983j

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.

to promote viral replication, while G 0 /G 1 or G 2 /M synchronization inhibits viral replication [10] . This indicates a possibility for developing cell cycle-modifying agents (especially those that induce G 0 /G 1 or G 2 /M arrest) as a novel strategy to treat HFMD.

Natural products have played pivotal roles in the drug discovery and development process. It has been noted that natural products embody more ''privileged structures'' [11] than purely synthetic chemical libraries and possess the characteristics and therapeutic effects of Chinese herbs [12] . Natural product-based drug discovery research programs represent important avenues for drug development [13] , including antiviral drugs [14, 15] . For example, emodin (1,3,8-tri-hydroxy-6-methylanthraquinone) is an active ingredient sourced from herbs belonging to genera Rheum, Polygonum, Rhamnus and Senna. Extracts from these herbs have been used for the treatment of epidemic-prone diseases [16] . Emodin has been shown to inhibit the replication of severe acute respiratory syndrome (SARS) virus [17] , hepatitis B (HBV) [18] , and herpes simplex viruses (HSV) [19] .

Artemisinin, a drug obtained from the plant Artemisia annua, is recommended by the WHO for the treatment of infections, such as drug-resistant Plasmodium falciparum strains, cerebral malaria and malaria in children [20] . A major saponin of this herb, astragaloside, has been shown to possess anti-cancer, anti-fatigue, anti-coxsackie B virus, and anti-inflammatory effects [21] . However, to date, its anti-EV71 activity has not been well investigated.

In the present study, we aimed to elucidate the anti-EV71 activity of the natural active components from Rheum emodin, Astragaloside and Artemisinin. We observed that Rheum emodin protects host cells against EV71 infection, decreases viral genomic levels and viral protein expressions, and decreases EV71 virulence by intervening in the cell cycle environment that favors viral replication; however, Astragaloside and Artemisinin did not show similar abilities. These results reinforce our model of the EV71 viral pathogenic mechanisms that require regulation of the cell cycle environment and suggest that Rheum emodin is a potential candidate for the treatment and prevention of HMDF disease.

The Changchun077 strain of EV71 has been reported previously [1] . This virus strain was propagated in African green monkey kidney cells (Vero). Vero (No CCL-81) and MRC5 (No CCL-171) were purchased from the ATCC (Manassas, VA, USA) and were maintained in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA).

Viral titers were determined by measuring the TCID50 in a microtitration assay using Vero cells, as described previously [22] . Briefly, Vero cells were seeded onto 96-well plates and incubated at 37 °C for 24 h. Virus-containing supernatant was serially diluted 10-fold and 100 µL was added per well in octuplicate. The cytopathic effects were observed once per day until the experimental endpoint was reached. The TCID50 was determined by the Reed-Muench method [23] , which is based on the assumption that 1×10 5 TCID50/mL will produce 0.7×10 5 plaque forming units/mL (www.protocol-online.org/ biology-forums/posts/1664.html).

Cells were mock-infected or infected with EV71 at an MOI of 1 for 2 h. Aafter 2 h of virus adsorption, cells were washed with PBS once and then fresh culture medium was added.

Cell growth inhibition test Inhibition of cell growth was determined by MTT (Sigma, St Louis, MO, USA) assay. MRC5 cells (1.0×10 4 cells/well) were seeded onto 96-well culture plates (Nunc, Roskilde, Denmark). After a 24 h incubation, different concentrations of Rheum emodin (Rheu), Artemisinin (Arte) and Astragaloside (Astra), which were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), were added individually to the plates. Following incubation, cell growth was measured at 48 h by the addition of 20 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL) at 37 °C for 2 h; DMSO (150 µL) was added to dissolve the formazan crystals. Absorbance was measured at 492 nm with an enzyme-linked immunosorbent assay plate (ELISA) reader (Bio-Rad, Hercules, CA, USA). The percentage of inhibition was calculated as follows: Inhibitory ratio (%)= [A 492 (control)-A 492 (sample)]/[A 492 (control)-A 492 (blank)]×100%.

For observing the cytopathic effects, cells were grown on a culture dish and infected by EV71 for 24 h. The morphological changes were observed and microscopic photographs were taken (Olympus, Tokyo, Japan).

The nuclear stain Hoechst 33258 (Sigma, St Louis, MO, USA) was used to visualize nuclear changes by fluorescence microscopy. Briefly, MRC5 cells were plated onto 6-well plastic culture dishes (4×10 5 cells/well) and infected with EV71 for 2 h; treated with drugs for 22 h; washed with PBS and fixed in 3.7% formaldehyde for 1 h; washed with PBS and then stained with 5 mg/L Hoechst 33258 for 30 min. Nuclear changes were observed under a fluorescence microscope at an excitation wavelength of 350 nm with an emission filter at 460 nm (Leica, Nussloch, Germany).

Cell cycle analysis by flow cytometry Nuclear DNA content was measured using propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS). Adherent cells were collected by treatment with trypsin and then washed with phosphate-buffered saline (PBS). Cells 

To analyze the virus present in the supernatant, intracellular virus and total virus were made up to 5 mL with DMEM medium and subjected to repeated freezing and thawing for three cycles. Subsequently, these samples were centrifuged at 3000 rounds per minute for 20 min and then the supernatant containing the virus was collected. Intracellular virus and total virus were obtained by centrifugation at 30 000 rounds per minute for three hours with 30% sucrose using a type 70.1 Ti Rotor in a Beckman ultracentrifuge (Optimal L-100XP ultracentrifuge Beckman Coulter). The samples were then collected for Western blot analysis. For determining cyclin A2 and CDK 2 expression, cells were collected at 36 h after EV71 infection and lysed directly in sodium dodecyl sulfate (SDS) sample buffer [60 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue], followed by boiling for 10 min. Whole-cell lysates were further subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and detected with the corresponding primary and alkaline phosphatase-conjugated secondary antibodies. The membranes were then reacted with 5-bromo-4-chloro-39-indolylphosphate (BCIP) and nitro-blue tetrazolium (NBT) substrate (Sigma). The anti-CDK 2 (Boster) and anti-cyclin A2 (Proteintech) mouse or rabbit antibodies were used in the Western blot analyses. Anti-VP1 polyclonal antibodies from rabbit were prepared in our laboratory. Rabbit or mouse secondary antibodies were obtained from Proteintech. The intensity of the bands was calculated by ImageJ (NIH ImageJ), and the value of the Con/Drug refers to the intensity of the VP1 protein band from the control group/the intensity of the VP1 protein band of the drug treatment group in the supernatant, intracellular and total virus. Quantitative real-time PCR RNA was extracted from infected cells treated control medium and drugs using the Trizol reagent (Gibco-BRL, Rockville, MD, USA) and isolated as specified by the manufacturer. The RNA was treated with DNAse (DNase I-RNase-Free, Ambion) to remove any contaminating DNA. Then, 200 ng of total RNA was reverse-transcribed with oligo dT primers using the High Capacity cDNA RT Kit (Applied Biosystems) in a 20 µL cDNA reaction as specified by the manufacturer. For quantitative PCR, the template cDNA was added to a 20 µL reaction with SYBR GREEN PCR Master Mix (Applied Biosystems) and 0.2 µmol/L of primers for VP1 and GAPDH.

The forward and reverse primer sequences for VP1 are 5'-AGCACCCACAGGCCAGAACACAC-3' and 5'-AT CCC-GCCCTACTGAAGAAACTA-3' and those for GAPDH are 5'-GCAAATTCCATGGCACCGT-3' and 5'-TCGCCCCA CTT-GA TTTTGG-3'.

Amplification was carried out using an ABI Prism 7000 for 40 cycles with the following conditions: an initial denaturation at 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min; then 1 cycle of 95 °C for 1 min, 55 °C for 30 s and 95 °C for 30 s. The fold changes were calculated relative to GAPDH using the ∆∆Ct method for VP1-coding sequence analysis.

Data are presented as the mean±standard deviation (SD). Between-group differences were assessed using Student's t-test. P values of <0.05 were considered statistically significant.

To prove that the anti-virus effects of Rheum emodin, Artemisinin and Astragaloside in MRC5 cells are not due to their cytotoxicity, MRC5 cells were treated with Rheum emodin, Artemisinin or Astragaloside at various doses to confirming their ability to inhibit cell growth. MRC5 are human lung fibroblast cells. Although EV71 infection mainly leads to central nervous system symptoms, the lungs can also be infected by the EV71 virus in vivo [24] , meaning that MRC5 cells can be used as a cell model. The cell inhibitory ratio was detected via MTT analysis at 48 h post-treatment. After 48 h of treatment with 0, 13.69, 27.38, 54.77, 82.15, 109.53, and 136.92 µmol/L of Rheum emodin, the concentrations with inhibitory ratios below 10% ranged from 0 to 82.15 µmol/L ( Figure 1A ). After 0, 1, 5, 25, 50, 75, and 85 µmol/L of Artemisinin or Astragaloside treatment for 48 h, the concentrations of Artemisinin with inhibitory ratios below 10% ranged from 0 to 75 µmol/L ( Figure 1B ) and the concentration of Astragaloside with inhibitory ratios below 10% ranged from 0 to 85 µmol/L ( Figure 1C) . Therefore, the range of concentrations for Rheum emodin (0 to 82.15 µmol/L), Artemisinin (0 to 75 µmol/L) and Astragaloside (0 to 85 µmol/L) did not affect cell survival. Based on the results of both a published study [25] [26] [27] and our MTT assay, we chose the following concentrations for further study: 29.60 µmol/L of Rheum emodin, 50 µmol/L of Artemisinin or 50 µmol/L of Astragaloside for anti-EV71 activity. Figure 1D illustrates the flow diagram of how to treat MRC5 cells in the designed experiment.

To further exclude the possibility that the anti-viral effects of Rheum emodin, Artemisinin or Astragaloside was due to their cytotoxic effects, as well as to confirm their protective effect on host cells, 29.6 µmol/L of Rheum emodin, 50 µmol/L of Artemisinin and 50 µmol/L of Astragaloside were chosen based on the previous results to treat mock-infected and infected cells for 22 h. There were no effects observed for either cell viability or morphology in the infected cells ( Figure  2A2 , 2A3, and 2A4) when compared with mock-infected cells ( Figure 2A1 ). Meanwhile, it was observed that numerous cells were rounded up and detached from the bottom of the dish in the cell morphological analysis ( Figure 2B1 ) compared to the mock-infected cells at 24 h post-infection of EV71 infection at a MOI of 1 ( Figure 2A1 ). While Rheum emodin treatment ( Figure 2B2 ) inhibited cell morphologic changes including being rounded up and detached from the bottom of the dish due to EV71 infection, Artemisinin ( Figure 2B3 ) and Astragaloside ( Figure 2B4 ) did not. Nuclear staining tests were also completed to confirm the effects of Rheum emodin, Artemisinin and Astragaloside on cell death. After the EV71-infected cell nuclei became condensed and bright ( Figure 2C1 ), which indicated cell death, Rheum emodin treatment inhibited the appearance of condensed and bright nuclei induced by EV71 infection (Figure 2C2 ), while neither Artemisinin ( Figure 2C3 ) nor Astragaloside inhibited cell death induced by EV71 infection ( Figure 2C4 ). Therefore, Rheum emodin possessed the ability to protect host cells from EV71 viral damage, while Artemisinin or Astragaloside were unable to achieve the same results.

Effect of Rheum emodin, Artemisinin and Astragaloside on viral genomic RNA replication To examine whether viral RNA replication and/or tran-scription was influenced by treatment with these drugs, we assessed EV71 VP1-coding sequence levels by qRT PCR at 12 h post-infection (EV71 has a single-stranded, positive-sense RNA genome, so detecting the VP1-coding sequence level is equivalent to detecting the level of the EV71 genome). EV71 RNA levels in Rheum emodin-treated cells were significantly lower than those in the control cells (15.75%±1.81% of control cells, Figure 3A Figure 4) . Thus, only Rheum emodin persistently inhibited the expression of viral proteins, while Artemisinin or Astragaloside did not show a sustained inhibitory effect on viral protein expression over an extended period of time.

To understand the inhibitory effect of the drugs on EV71 production, Viral TCID50 was examined at 36 h post-infection to evaluate the viral virulence. The TCID50/mL was found to be 3.16×10 9 in the mock-treated cells and 2.37×10 2 in the Rheum emodin-treated cells. The TCID50/mL remained at 3.16×10 9 in both the Artemisinin-and Astragaloside-treated cells (Table  1) . Furthermore, we also observed a 1.33×10 7 -fold increase in the TCID50/mL of mock-treated cells over the Rheum emodin-treated cells (Table 2) . Thus, Artemisinin and Astragaloside did not inhibit viral production, while Rheum emodin exhibited a significant inhibitory effect on viral reproduction.

In our previous study, we found that the EV71 virus induced cell cycle arrest at S phase to promote viral replication and that G 0 /G 1 or G 2 /M synchronization inhibited viral replication. Therefore, we examined whether the effects of emodin Figure 5B ). Artemisinin treatment decreased S phase from 54.08%±1.24% to 47.65%±0.23% (P<0.01) after EV71 infection ( Figure 5D ); Astragaloside treatment also induced a decrease from 50.74%±1.13% to 46.01%±1.16% (P<0.01) post-EV71 infection ( Figure 5F ). To further emphasize the effects of Rheum emodin, Artemisinin and Astragaloside treatment on S phase in EV71-infected cells, we analyzed the percentage of S phase cells after EV71 infection. The percentage of S phase cells after Rheum emodin treatment was 55.78% of that observed in the mock-treated cells ( Figure 5C ). The corresponding S phase percentages in the Astragaloside-and Artemisinin-treated cells were 90.68% ( Figure 5E ) and 88.11% of mock-treated cells ( Figure 5G ), respectively. Thus, our findings suggest that Rheum emodin inhibits the S-phase accumulation induced by EV71 infection.

Western blotting was performed to observe the expression of CDK 2 and cyclin A2 at 36 h post-infection; this time point was chosen according to our previous study [28] . A significant decrease in CDK 2 and cyclin A2 was observed in the viralinfected cells compared to the mock-infected cells. Rheum emodin further decreased the expression of CDK 2 and increased the expression of cyclin A2 in EV71-infected cells ( Figure 6A ). This effect was not observed with either Artemisinin ( Figure 6B ) or Astragaloside treatment ( Figure 6C ). Therefore, Rheum emodin appeared to inhibit the manipulation of the host cell cycle by EV71 virus by regulating CDK 2 and cyclin A2 expression.

Human enterovirus 71 (EV71) is the primary causative agent Infected, infected EV71; con, control; Rheu, Rheum emodin; Arte, Artemisinin; Astra, Astragaloside Table 2 .

The fold of TCID50/mL 1.33×10 7 1 1

Infected, infected EV71; con, control; Rheu, Rheum emodin; Arte, Artemisinin; Astra, Astragaloside for HFMD outbreaks in Asian countries [1, 2, 5, 9] . Development of EV71 viral vaccines [29, 30] and inhibitors that target various phases of the EV71 lifecycle is currently at a preclinical stage [31] . Currently available antiviral drugs have limited efficacy against EV71 infection. Chinese herbs have been used for the prevention and treatment of epidemic diseases since ancient times. In this study, we investigated the effect of three natural ingredients, Rheum emodin, Astragaloside and Artemisinin, which are extracted from Chinese herbs on EV71 viral replication.

Cell death led to the loss of function in damaged cells; for example, neural cell death will lead to amnesia, mental disorder and other conditions [32] and muscle cell death will lead to outcomes such as paralysis or myasthenia [33] . Cell death also leads to the release of cellular contents and eventually to inflammation. Many studies have been completed in vivo and in vitro to identify the inflammatory factor released after cell death [34] [35] [36] . In this study, we found that Rheum emodin has the ability to protect host cells from the damage associated with EV71 viral infection. Therefore, Rheum emodin inhibits cell death caused by EV71 virus infection, which ultimately inhibits the loss of host function and inflammatory occurrence.

Cell death from viral infection is usually due to viral replication. Hence, we investigated whether Rheum emodin inhibited viral replication. Our results clearly indicate that Rheum emodin decreased the levels of viral genomic level, viral protein expression, and EV71 virulence compared to the control-treated group. Therefore, Rheum emodin inhibits cell death by inhibiting viral production. The fold difference in the TCID50/mL, mRNA levels, and protein expression between the control-treated and Rheum emodin-treated cells [37] , but not Artemisia carvifolia or Astragalus.

In the previous study, we showed that EV71 induced cell cycle arrest in S phase to facilitate viral production [10] . However, Rheum emodin decreased the amount of S phase favored by EV71 to increase the ratio of G 0 /G 1 phase compared with . Control medium (Con) and 50 µmol/L of Astragaloside (Astra) treatment for 22 h (F). The S phase percentage was compared between the control medium (Con) and the drug treatment groups in the EV71-infected (Infected) MRC5 cells in (C, E, and G). Control medium (Con) and 29.60 µmol/L of Rheum emodin treatment (Rheu) for 10 h (C). Control medium (Con) and 50 µmol/L of Artemisinin (Arte) treatment for 10 h (E). Control medium (Con) and 50 µmol/L of Astragaloside (Astra) treatment for 10 h (G). The results indicate the mean±SD of one experiment and are representative of three independent experiments. ** P<0.01.

the control treatment in this study. Additionally, the expression of CDK 2 and cyclin A2 further demonstrated the role of Rheum emodin in cell cycle regulation. It was therefore concluded that Rheum emodin inhibited EV71 viral replication by intervening in the cell cycle environment favored by the virus. Although Artemisinin and Astragaloside also displayed the ability to decrease the amount of S phase favored by the EV71 virus, the effect was not as potent as Rheum emodin. Indeed, EV71 appeared to overcome the inhibitory effect of Artemisinin and Astragaloside over 26 h.

In a previous study, exogenous expression of 3D, an RNAdependent RNA polymerase, was shown to mediate cell cycle arrest. Therefore, it is speculated that Rheum emodin might affect the function of 3D in viral replication. To our knowledge, members of the Flaviviridae family, which include hepatitis C (HCV) [38] , dengue virus [2, 39] , and classic swine fever [40] , also induce S-phase arrest. Notably, Picornaviridae and Flaviviridae share the common characteristic of being singlestranded, positive-sense RNA viruses. Therefore, Rheum emodin may be effective against single-stranded, positive-sense RNA viruses that favor S phase.

These results further prove our understanding of the EV71 viral pathogenic mechanisms and indicate that Rheum emodin is a potential candidate for the treatment and prevention of HMDF disease. Figure 6 . Western blotting shows the effects of Rheum emodin, Artemisinin and Astragaloside on expression of host cell cycle regulatory proteins after EV71 infection. MRC5 cells were mock-infected or infected with EV71 at a MOI of 1 for 2 h. Two hours later, the cells were treated with drugs that lasted for 34 h, and then the cells were collected for lysis. Cyclin A2 and CDK2 expression was detected by Western blot analysis. Control medium and 29.60 µmol/L of Rheum emodin (Rheu) treatment for 34 h (A). Control medium and 50 µmol/L of Artemisinin (Arte) treatment for 34 h (B). Control medium and 50 µmol/L of Astragaloside (Astra) treatment for 34 h (C). Histone or Tubulin was shown as a loading control. The results are representative of three independent experiments.

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Jing-hua YU designed the experiments and wrote the paper; Ting ZHONG conducted the experiments; Li-ying ZHANG, Zeng-yan WANG, Yue WANG, Feng-mei SONG, and Ya-hong ZHANG analyzed the data and prepared the viruses, cells and reagents.