| Background and objective Respiratory syncytial virus (RSV ) is the most important viral agent of lower respiratory tract infection in infants and young children worldwide It is also a major role in sudden infant death syndrome(SIDS) and a leading pathogen of nosocomial infection and one of the causes being responsible for acute respiratory tract infection in adults Effective therapy and vaccines are not available at present RSV genome is a single strand of negative-sense RNA which genomic RNA sequences are clear now RSV replication involves a switch from the stop- start mode This results in the synthesis of a positive-sense replicative intermediate RNA(RI RNA) Integrity of some genes sequences in genomic RNA is needed for RSV replication RSV RI RNA would not synthesize and replication of RSV would be blocked if genomic RNA was cut in some regions It has been demonstrated that RSV replication was effectively inhibited by cut phosphodiester bond in NS2gene start region RSV genomic negative strand RNA is transcribed into 11 mRNAs and then translated into 11 viral proteins utilizing its own RNA dependent polymerase Synthesis of RSV proteins could be inhibited by cut its mRNAs on proper site DNAzyme/Deoxyribozyme is another novel molecular biological tool novel tool after the ribozyme DNAzyme consists of a 15 nt internal loop as its catalytic domain and two flanking substrate-recognition domains of seven to eight nt each which is complementary to substrate The RNA substrate is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue DNAzyme has been applied in fields such as virus infection diease, tumor, cardiovascular disease and genetic disease But there is no report about anti- respiratory syncytial virus by using DNAzyme In this study DNAzymes have been designed and synthesized to target the RSV genome and also to target the M2 mRNA at different site The overall objective of this study is to lay the groundwork for treating patients with RSV infection by using DNAzyme to target the RSV viral RNA replication in epithelial cells of respiratory tract Methods Based on Santoro's reports, we have designed three DNAzymes DZ595,DZ604 and RSV2UDZ(NS1-2)to target the RSVgenome at the start of the NS2 gene and three DNAzymes DZ8269,DZ8277and RSV2UDZ(M2) to cleavage the M2 mRNA Selection of DNAzymes cleavage activity was performed by cell free assay A monkey kidney cell line, Vero ,and a human tracheal epithelial cell line,9HTE were used In RSV infected cell culture system RSV was propagated in Vero cells and 9HTE cells was served as targets of RSV infection A substrate cleavage DNAzyme, DZ604 which was selected by cell free assay was used In RSV infected cell culture system Then cytopathic effect(CPE) inhibition assay, plaque inhibition assay, and MTT assay were performed separately to detect the anti-RSV activity, protective function for RSV infected 9HTE cells and cytotoxicity of DZ604 ELISA was used to detect the RSV yield in each different group of DZ604 treatments and electron microscope was used to observe the effects of DZ604 acting on ultrastructure change of 9HTE cells induced by RSV Results RSV NS1/2RNA substrate could be cleaved by All of DZ595, DZ604 and RSV2UDZ(NS1-2) Among them DZ604 had the highest cleavage activity RSV M2mRNA substrate could be cleaved by DZ8269 and DZ8277 but could not be cleaved by RSV2UDZ(M2) RSV NS1/2RNA substrate could not be cleaved by anti- RSV M2mRNA DNAzymes RSV M2mRNA substrate could not be cleaved by Anti-RSV genomic RNADNAzymes RSV infected cell culture system we established stable for utilization: The 9HTE cells mainly became swelling and falling off and few of them formed syncytium after infected by RSV The CPE occurred time and scale of CPE depended on multiplicity of infection(MOI) of RSV 9HTE cells M swelled and fell off quickly in 12 hours after infected by RSV at 10 and 1 MOI With MOI of RSV decreased,the CPE of 9HTE cells occurred mo...
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