In Vivo Efficacy And Antiviral Mechanism Of Broad-spectrum Inhibitors Of Coronavirus

Coronaviruses(CoVs)are enveloped ss positive RNA viruses,which belong to the members of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales.They are widely distributed among human,mammalian and other hosts and are able to spread across species.When infected with coronaviruses,hosts usually suffered from respiratory symptoms accompany with mental disease.HCoV-NL63,HCoV-229E,HCoV-OC43 and HCoV-HKU1 mainly cause mild respiratory symptoms.However,zoonotic SARS-CoV,MERS-CoV and SARS-CoV-2 outbreak in late 2019 may lead to lethal severe respiratory symptoms and pose great threaten to human life Therefor coronaviruses have attracted wide attention all over the world.However,more worrying,little is known about the replication and dynamic infection of coronavirus in vivo and there are no available specific antiviral drugs against coronavirus.In vivo efficacy of inhibitors of coronaviruses and their antiviral mechanisms still need to be verified.Animal models are indispensable for in vivo evaluation of antiviral drugs after in vitro screening.It is nonintuitive and takes a long course for evaluation in conventional assays.Operations associated with SARS-CoV,MERS-CoV and SARS-CoV-2 are risky and complex and must be manipulated in BSL-3 laboratories.Unlike those lethal viruses,assays related with HCoV-OC43 and MHV-1 can be conducted in BSL-2 labs.What's more,all those viruses belong to ? group and share similar genome structure.At the same time,they can replicate in respiratory system and central nervous system.Along with new detection techniques,correlated animal models maybe promising platform for evaluation of antiviral drugs.What's more,mechanism of anti-coronavirus inhibitors should be elucidated for clinical application and investigation of more inhibitorsIn this study,we established an optical BALB/c mouse model using recombinant HCoV-OC43 expressing Rluc combined with bioluminescence imaging technology.At the same time,HCoV-OC43 infection in IFN?R-/-mouse model which may lead to lung pathology was constructed.In addition,in vivo efficacy of broad-spectrum anti-coronavirus molecules was verified using above mouse models and the MHV-1 mouse model.Furthermore,the molecular mechanism of the broad-spectrum anti-coronavirus inhibitor emetine was elucidated.The principal results are as follows:1.Establishment and application of an optical mouse model associated with HCoV-OC43 infection.rOC43-ns2DelRluc infection caused neurological symptom and was fatal to suckling mice of different genetic backgrounds(BALB/c and C57BL/6).Replication of rOC43-ns2DelRluc could only be detected in brains and spinal cords of BALB/c mice using bioluminescence imaging technology.What's more,virus titer in brains and spinal cords correlated well with the Rluc readout,especially infection through intracranial injection.Chloroquine,a previously reported anti HCoV-OC43 drug,could inhibit virus replication and attenuate the signals.furthermore,we found that lycorine could also restrain the replication of HCoV-OC43 after screening several small molecules which showed broad-spectrum anti-coronavirus activity in vitro.In brief,this platform could be applied to monitor neurological symptom caused by coronavirus replication and to evaluate the efficacy of antiviral therapies to treat neuroinvasive HCoVs in living mice.2.Establishment and application of HCoV-OC43 infection in IFN?R-/-mouse.Replication of HCoV-OC43 could be detected in brains and lungs after infected intranasally with rOC43-ns2DelRluc in IFN?R-/-mice.Virus titer was higher in brains and lungs of IFN?R-/-mice than those of their parental C57BL/6 mice and peaked at 6 dpi followed by elimination of host immune system.At the same time,rOC43-ns2DelRluc infection caused significant pathological changes,including alveolar rupture and aggregation of neutrophils,which were verified by immunohistochemical analysis.On the contrary,replication of rOC43-ns2DelRluc in parental C57BL/6 mice was limited.Chloroquine,a previously reported anti HCoV-OC43 drug,could inhibit virus replication and lung pathology caused by rOC43-ns2DelRluc infection.What's more,the experiment assays proved that lycorine could also inhibit the replication of HCoV-OC43 and relieve lung pathology after screening several small molecules which showed broad-spectrum anti-coronavirus activity in vitro indicating that this platform could be used to monitor lung pathology and to evaluate the efficacy of antiviral therapies to relieve lung pathology caused by coronavirus replication.3.Application of MHV-1 infection in A/J mouse of in vivo evaluation of broad-spectrum anti-coronavirus inhibitors.MHV-1 infection in A/J mice model was applied to in vivo evaluation of small molecules inhibitors against coronavirus.According to previous report,replication of MHV-1 peaked in brains,lungs,livers and spleens of A/J mice at 3 days post-infection.So viral titer in such tissues of A/J mice treated with several broad-spectrum anti-coronavirus drugs was qualified and the result proved that emetine could inhibit MHV-1 replication.4.Molecular mechanism of emetine with broad-spectrum efficacy against coronavirus infection.According to the time-of-addition assay,emetine mainly inhibited coronavirus replication at early stage including attachment,entry and synthesis of viral genome and protein.The experiment associated with specific steps of the viral life cycle proved that emetine could block entry of MERS-CoV and HCoV-NL63 into cells and broadly restrain genome replication without effect on attachment.What's more,membrane fusion assay suggested that emetine blocked MERS-CoV entry through blocking membrane fusion induced by S protein.In addition,transcription of viral genome was inhibited by emetine when binding to ribosome and proteases related with replication complex were reduced.That might be the molecular mechanism why emetine showed broad-spectrum anti-coronavirus activityAbove all,rOC43-ns2DelRluc infection in BALB/c mice or IFN?R-/-mice could be applied to monitor neurological symptom or lung pathology caused by coronavirus replication and to evaluate the efficacy of antiviral therapies to treat HCoVs.In vivo efficacy of small molecules(Chloroquine,Lycorine,Emetine)with broad-spectrum anti-coronavirus activity were verified using above two mouse models and the MHV-1 mouse model.New molecular mechanism and target of emetine with broad-spectrum efficacy was elucidated.