Mechanism Of Andrographolide Inhibiting The Replication Of Enterovirus D68

Backgrounds:The genus Enterovirus(EV)of the family Picornaviridae includes several medically important pathogens such as poliovirus,echoviruses,coxsackieviruses,numbered enteroviruses,and rhinoviruses.In past decades,several enteroviruses have emerged as serious public health concerns.For example,poliovirus,the pathogen that causes acute spinal cord paralysis in humans;EV-A71,the pathogen that causes hand-foot-mouth disease in children especially in Asia.Recently,enterovirus D68(EV-D68;originally classified as human rhinovirus87),caused a large outbreak of severe lower respiratory tract disease in North America.EV-D68 was first isolated in California from patients with pneumonia and bronchiolitis in 1962 and has since been infrequently reported in association with a wide range of clinical manifestations including mild-to-severe respiratory disease.In 2014,the largest outbreak of EV-D68 infection occurred in the United States.Most patients were children with severe respiratory conditions that necessitated intensive care.Patients with a history of asthma or reactive airway disease were more likely to require ventilator support.Since the 2014 outbreak,an increasing number of clusters of EV-D68 infection have been reported in Europe,Asia,and Oceania.EV-D68 has also been implicated in polio-like neurological disorders such as acute flaccid myelitis.Despite the spread of EV-D68 viruses,there are no effective vaccines or antiviral agents available for clinical use.It was recently reported that three antiviral drugs in clinical trials for enteroviral infections(pleconaril,pocapavir,and vapendavir)failed to inhibit EV-D68 infection.Andrographis paniculata Nees has been traditionally used for centuries in Asia to treat various diseases such as respiratory infection,fever,diarrhea,and bacterial dysentery.Andrographolide(ADO)is a bicyclic diterpenoid lactone and is thought to be the major bioactive component of A.paniculata,exerting anti-inflammatory,anti-cancer,antiviral and immune-modulating activities.Previous studies have shown that ADO has activity against several viruses including the human immunodeficiency virus,hepatitis B virus,dengue virus,and herpes simplex virus.However,the antiviral mechanism of andrographolide remains to be explained.In this study,we evaluated the activity of ADO against the EV-D68 virus.Our results revealed the antiviral activity of ADO and explored its possible mechanisms and shed light on its utility for the treatment of EV-D68 infections.Aim: In this study,we found that andrographolide(ADO)exhibited potent resistance to EV-D68 infection.ADO significantly inhibited viral replication and protein synthesis without significant cytotoxicity.We focused on the various steps of the EV-D68 life cycle and aimed to find possible targets for ADO to inhibit,providing a a new idea for the optimization and development of new antiviral drugs.Methods:(1)The effect of ADO on EV-D68.1)Cytopathic effects(CPEs): The ADO-treatment group and control group were infected with the same dose of virus,and the degree of CPEs was observed 48 hpi and photographed.2)Effect on viral RNA replication: ADO pretreated cells and control groups were infected with the same dose of virus,cells were harvested at specific time points,viral RNA was extracted,and the effect of ADO on viral RNA replication was detected by q RT-PCR.3)Effects on viral protein synthesis: ADO pretreated and control cells were infected with the same dose of virus,and effects of ADO on viral protein synthesis were detected by western blot and immunofluorescence.4)Effect on the titer of progeny virus: ADO pretreated cells and control group were infected with the same dose of virus,and the supernatant was harvested 48 hpi,and the cells were infected by gradient dilution in 96-well plates.The cells were observed after 5-7 days of incubation.We used the Reed-Muench method to calculate virus titer.(2)Study on the mechanism of ADO inhibiting EV-D68 virus 1)The effect of ADO on the attachment and entry step of the virus: set the virus in attachment and entry phase through specific experimental conditions,extract viral RNA,and detect RNA level by q RT-PCR.2)Effect of ADO on viral transcriptional activity: we constructed the EV-D68 5’UTR luciferase reporter system,and the 5’UTR luciferase plasmid alone or in combination with the viral 3D protein(RNA-dependent RNA polymerase)expression plasmid were transfected into ADO pretreated or control 293 T cells.The luciferase level was detected 48 h post transcription.3)Effect of ADO on immune response induced by virus: the m RNA level of interferon-βand interferon-inducible gene-56 was detected by q RT-PCR.4)Effect of ADO on the acidification process of virus-containing endosomes: after incubation with an acid-sensitive green fluorescent dye and virus,the ADO and DMSO pretreated cells were infected.Laser confocal microscopy was used to detect the difference in green fluorescence.(3)ADO’s half effective concentration and cytotoxicity determination.1)Detection of half effective concentration of ADO: pretreat cells with different concentrations(0,5,10,20,40μM)of ADO and harvest cells 24 hpi.q RT-PCR was used to detect viral RNA levels.We draw a curve and calculate the EC50.2)Cytotoxicity determination of ADO: cells were treated with ADO at various concentrations(0,2.5,5,10,20,40,80,100 and 160μM),and the highest concentration of DMSO was used as a positive control.Cell viability was measured by MTS method after 24 h incubation.Resluts:(1)ADO has a significant inhibitory effect on EV-D68 virus.The degree of CPE in ADO treated cells was significantly reduced.Through Qrt-PCR,western blot,immunofluorescence and progeny virus titer detection,it was found that ADO can reduce the replication of viral RNA replication and protein synthesis.Both the Fermon and circulating strains(MO and KY)were potently inhibited by ADO.(2)Study on the mechanism of ADO inhibiting EV-D68 virus.In the attachment and entry experiments,we examined the viral RNA levels respectively and found no obvious difference between two groups,indicating that ADO does not target on the viral capsid protein or the viral receptors.In the luciferase reporter assay system,we found that viral RNA dependent RNA polymerase 3D protein has a significant potentiating effect on viral 5’UTR activity,which is consistent with its function.The results showed that whether ADO treated or not,no difference was detected on the 3D protein-dependent 5’UTR activity.In addition,we measured the m RNA levels of interferon-β and interferon-induced gene-56 by q RT-PCR.There was no significant difference between the two groups,indicating that ADO had no influence on immune response.Then we use a p H-sensitive dye(which can be stimulated by green fluorescence when the p H is at 5-8,known as the endosomal vesicle acidic range)to incubate with virus and then infect the cells.The fluorescence of endosomal vesicles was observed by confocal microscopy at specific time points.The fluorescence intensity in the control group reached a peak at 2hpi and the signal got decayed rapidly afterwards.In the ADO treated cells,there was no fluorescence peak within the first several hours after infection.And the fluorescence intensity was very weak.Similarly,the same phenomenon was found in the enterovirus-A71.The results suggested that the target of ADO was very much likely to be the viral endosomal vesicle.By interfering with the acidification and maturation process of vesicles,ADO can not complete the decapsidation of the viral particles and the release of viral genome into the cytoplasm,so that it can not participate in the effective proliferation.Finally we examined the effect of ADO on the EV-D68 epidemic strains(MO and KY),and found that there was also an obvious inhibitory effect.The drug can protect cells and present with a reduction of CPEs,and decrease the viral RNA andprotein levels.(3)ADO’s half effective concentration and cytotoxicity determination.The half effective concentration of ADO on different strains: EC50(Fermon)=3.45μM;EC50(MO)=4.1875μM;EC50(KY)=5.807μM.The cytotoxicity of ADO: CC50 = 75 μM(taking RD cells as an example).It can be seen that ADO can effectively inhibit EV-D68 virus in a relatively safe concentration to host cells.Conclusion:(1)ADO has significant inhibitory effects on Enterovirus D68.(2)ADO inhibits viral uncoating process by blocking the acidification and maturation process of viral endocytic vesicles,thus reducing the number of viruses that can be efficiently replicated.(3)Andrographolide also applies to enterovirus 71 by interfering with the endosome acidification and maturation process,indicating that this mechanism is universally applicable and maybe particularly effective for viruses that require low p H to complete the uncoating process.(4)Andrographolide also possesses a significant inhibitory effect on the epidemic strains(MO,KY),and it establishes itself as a candidate for the prevalence of EV-D68.