Construction Of Chikungunya Virus Infectious Clones And Preliminary Studies On Their Pathogenesis

BackgroundChikungunya virus(CHIKV)is a mosquito-borne Alphavirus of the Togaviridae family.It causes Chikungunya fever(CHIKF),which is characterized by fever,rash,and joint pain.In recent years,with the global climate and environmental factors changing,human activities and migration,the transmission range of CHIKV has been expanding.In particular,after the outbreak of the Eastern/Central/Southern African(ECSA)Chikungunya virus in 2004,it evolved into the Indian Ocean Lineage(IOL),and CHIKV acquired an adaptive mutation(A226V),which increased fitness of CHIKV in Ae.albopictus and improved transmissibility to vertebrate species,resulting in a widespread epidemic in Asia.In the past two decades,CHIKV has spread rapidly from Africa to Asia,America and Europe,causing millions of infections and thousands of deaths.It has become a global public health problem.So far there are no approved human vaccines or antiviral drugs.Objective(1)To identify the differences in cell invasion,replication and pathogenicity among different strains of CHIKV,and identify the key proteins and amino acid residues that cause the pathogenicity differences of CHIKV;(2)To explore the mechanism of virus gene mutations affecting CHIKV cell invasion,replication and pathogenesis from both cell and animal models,so as to provide new targets for the development of CHIKV vaccine and antiviral drugs.Methods(1)According to Gen Bank,the full-length genome of ECSA type(Ross,Ross-low-psg)and IOL branch(LR2006-OPY1)was synthesized in several fragments.A T7 promoter was added into the 5'UTR,and a poly A structure,followed by a Not?restriction enzyme site,was added into the 3'UTR.The whole sequence was inserted into the p UC18 vector.The virus protein was expressed in 293T cells by transfection,and the virus was rescued by infecting Vero cells with transfected supernatant.(2)Using reverse genetics,we inserted the enhanced green fluorescent protein(Egfp)gene into CHIKV between ns P4 and C gene.Then we infected Vero cells with CHIKV-Egfp.We can easily judge the replication efficiency of virus in cells according to the change of fluorescence intensity and area,and compare the replication dynamics differences among different strains.(3)Using the same method,Nano Luc~?luciferase reporter gene was inserted into CHIKV.Then we infected mice with CHIKV-Nano Luc.Infected and mock-inoculated mice were intraperitoneally injected with furimazine and anesthetized for 8 min.Then,acquire images.(4)The structural genes of Ross strain and LR2006 strain were exchanged,in order to observe the change of the plaque size of the recombinant virus and its parent virus after replacement;the ns P1-4 and E1-3 genes of Ross strain were replaced with the corresponding genes of Ross-low strain respectively to compare the change of virulence of the recombinant virus.Results(1)Established the reverse genetic technology of CHIKV,obtained the infectious clone of CHIKV,and rescued the Ross strain,Ross-low-psg strain,LR2006-OPY1 strain and CHIKV expressed Egfp reporter genes(Ross-Egfp,LR-Egfp),or Nanoluc?luciferase reporter genes(Ross-Nano Luc,LR-Nano Luc).Ross strain with LR2006 structural gene replacement(Ross-LR),and LR2006 with Ross structural gene replacement(LR-Ross),and Ross strain with Ross-Low gene replacements(Ross-E1,Ross-E2,Ross-E3,Ross-NS2,Ross-NS3,and Ross-NS4).(2)Compared with the parent virus,the virus containing the Egfp reporter gene had no significant difference in cytopathic effect,virus plaque size,and mouse pathogenicity.(3)Ross-Egfp and LR-Egfp infected Vero cells.About 12 hours after infection,LR-Egfp-infected cells began to show fluorescence.About 18 hours after infection,Ross-Egfp-infected cells began to show fluorescence.In the plaque experiment,LR2006 strain formed a larger virus plaque 48 hours after infection,while Ross strain formed a relatively small virus plaque.After replacing the structural genes of the two strains,LR-Ross formed smaller plaques than the parent LR2006,while Ross-LR formed larger plaques than the parent Ross strain,and compared with the LR2006 strain.Plaques are the same size.The above results indicate that CHIKV structural protein affects the process of virus invasion and replication in cells.(4)Rescue virus was used to establish a mouse model.Ross-Nano Luc and LR-Nano Luc were nasally inoculated into 6-week-old C57BL/6 mice.The mice in the Ross group got sick on the 7th day.Luminous signals appeared in the brain,and no luminous signals were observed in the mouse brain in the LR2006 group.After that,the mice in the Ross group showed obvious symptoms of central nervous system infection and died.The LR2006 group had mild symptoms,only the weight loss,and no central nervous system symptoms.The above results show that Ross strain is more likely to invade the central nervous system of mice,is neurotropic,and is also the main cause of death in mice.(5)Ross strain and Ross-Low strain were nasally inoculated into 6-week-old C57BL/6mice.Both groups of mice showed weight loss 7 days after infection,and Ross group suffered infection after infection.Death occurred on the 9th day.There were no obvious central nervous system symptoms in the Ross-Low group,and the body weight gradually began to recover.The survival rate of mice in Ross group and Ross-Low group within 14days was 50%and 100%,respectively.The survival curves were significantly different(P<0.05).(6)Various types of Ross mutations and Ross and Ross-Low infected Vero cells,respectively.Among them,Ross-E2 produced large plaques different from the parent Ross strain,and the remaining Ross-E1,E3,NS2,NS3,and NS4 were all small plaques.In animal experiments,6-week-old C57BL/6 mice were nasally inoculated,and the toxicity of Ross-E2 and NS3 was reduced.The 14-day survival rate was 100%.E2 group was not seen obvious symptoms and weight trends.There were significant differences from the Ross group(P<0.05).Although some of the other mutations had decreased virulence,they still had central nervous system symptoms and mice died.The above results indicate that the CHIKV E2 protein affects the replication efficiency of the virus in the cells,and at the same time has a key role in the pathogenicity of mice,especially the neuroinvasiveness.ConclusionsIn summary,this project established CHIKV infectious clone,constructed a mouse model of CHIKV infection,especially in nervous system invasion,and preliminarily determined the key influence of E2 protein on virus invasion cells,replication and pathogenicity in mice.It is of great significance for developing vaccines and drugs against CHIKV infection and disease.