The Mechanism Of Pyrimidine Analog FNC Potently Inhibits Multiple Enteroviruses

Enterovirus belongs to picornavirus family whose genetic is a single-stranded positive-strand RNA.Enteroviruses cause a wide range of diseases,such as hand,foot and mouth disease?HFMD?,encephalitis,myocarditis,acute flaccid myelitis?AFM?,pneumonia,and bronchiolitis.Therefore,broad-spectrum anti-enterovirus drugs are urgently needed to treat enterovirus infection.FNC?2'-fluoro-4'azido-cytidine?,a novel small nucleoside analogue,has been demonstrated to be a potent inhibitor of HIV,HCV and HBV.It has entered into a clinical phase II trial in China for HIV infection treatment.Previous studies revealed that FNC inhibited HCV replication through preventing viral RNA replication events by targeting NS5B,the RdRP encoded by HCV.However,it has not been reported that FNC inhibits enterovirus replication through the RNA-dependent RNA polymerase.Firstly,we conducted in vitro experiments.We found that FNC can effectively inhibit the replication of various enteroviruses including EV71,CA16,CA6,EVD68,and CVB3 at the nanomolar level.FNC was used to study the antiviral effects of the above five enteroviruses.We investigated the enteroviruses expression levels of RNA and protein and the content of enteroviruses particles in the supernatant.It was initially determined that FNC can effectively inhibit the replication of a variety of enteroviruses.However,FNC cannot inhibit the replication of respiratory syncytial virus and influenza A virus in vitro.So we speculate that the inhibitory effect of FNC on enteroviruses is specific.Since the genome RNA of enteroviruses replicate via RNA-dependent RNA polymerase(called 3D^pol protein),we speculate that FNC may target 3D^pol to specifically inhibit enterovirus replication.We observed that FNC inhibited both positive-and negative-strand enteroviruses RNA synthesis,including that for EV71,CA16,CA6,CVB3,and EVD68,by RT-qPCR detecting positive-or negative-strand enteroviruses RNA after incubation with the tested concentrations of FNC.For EV71,CA16 and CA6,FNC showed stronger inhibition against positive-strand than negative-strand RNA synthesis.However,the inhibitory effect of FNC on CBV3 and EVD68was the opposite.Furthermore,we constructed and purified the prokaryotic expression plasmid of EV71-3D^pol.The purified EV71-3D^pol for isothermal titration?ITC?experiments,and the results showed that FNC and EV71 3D^pol have a direct interaction(Kd=6×10^-6 mol/L).Through cell experiments and ITC experiments,FNC inhibits positive-and negative-strand RNA synthesis of enteroviruses by interacting and interfering with the activity of EV71viral RNA-dependent RNA polymerase(3D^pol).Secondly,we conducted in vivo experiments.In a previous study,we established a lethal EV71 and CA16 infection neonatal mouse model.Here,we investigated whether FNC protects neonatal mice against lethal challenge by EV71 and CA16 viruses.We intracerebrally injected Changchun-circulating EV71 CC063 and CA16 CC045 into one-day-old mice,followed by FNC or DMSO treatment,and we carried out drug intervention to explore whether FNC has a protective effect on neonatal rats.The results showed that mice infected with EV71 CC063and CA16 CC045 became sick on day 5 and day 8 post infection and presented a gradual aggravation tropism,with a clinical score of grade 4 on day 6 for EV71 CC063 and a clinical score of grade 3 on day 12 for CA16 CC045.Compared to the FNC-treated mice,the DMSO-treated mice exhibited observable illness symptoms,such as single-limb paralysis,which occurred approximately 2 days earlier.At day 9 post EV71 challenge,the status of the DMSO-treated mice deteriorated,reaching a peak of death or symptoms such as four-limb paralysis,whereas mice with the FNC intervention presented fewer deaths or two-limb paralysis.At the same time,FNC treatment completely decreased the EV71 viral load in the spine skeletal muscle and hind-limb muscle and moderately reduced viral load in the brain and lung compared to those in the DMSO treatment group.In addition,FNC treatment completely decreased the CA16 viral load in all detected tissues compared to those in the DMSO treatment group.After that,tissues were sampled and histologically examined by H&E staining.EV71and CA16 infection both resulted in severe necrosis,including significant muscle bundle fracture,dissolution of muscle fibre cells in the spinal skeletal muscle and the hind-limb muscle of EV71 or CA16 infected mice.And CA16 but not EV71 infection caused obvious lung lesions including severe enlarged alveoli.Patchy vacuoles in the brain were observed.These results indicated definite damages on mice tissue caused by EV71 and CA16 infection.However,FNC treatment significantly reduced the damage to mouse tissues of EV71-infected spinal skeletal muscle,hind-limb muscle,and CA16-infected brain,spinal skeletal muscle and hind-limb muscle.Alleviated lesions were found in EV71-infected brain and CA16-infected lung.Importantly,the mortality due to EV71 and CA16 challenge reached 90%and 30%,respectively,while FNC treatment greatly reduced the mortality to 20%and 0%.FNC treatment significantly improved clinical manifestations and survival rates,indicating that FNC effectively protected against EV71 and CA16 challenge in vivo.In conclusion,through in vitro and in vivo experiments,we determined that FNC can effectively inhibit the replication of multiple enteroviruses,providing important clinical information for FNC as a broad-spectrum inhibitor of human enterovirus pathogens.