猫杯状病毒感染性克隆的构建及拯救病毒的鉴定OA

This study aimed to establish a reverse genetics system for feline calicivirus(FCV),investigate its pathogenic mechanisms based on this system,and provide a scientific basis for subse-quent vaccine development.Using FCV strain HZ2024 as the subject,a DNA-launched infectious clone sys-tem was constructed.Four fragments of the complete FCV genome were sequentially inserted into the pAY-CHS recombinant plasmid;the plasmid was transfected into F81 cells for passage and identifica-tion.Results showed:FCV HZ2024 gene fragments were amplified and inserted into the DNA-launched sys-tem plasmid,successfully constructing the DNA-launched system rescued recombinant virus rFCV HZ2024-RE.Typical cytopathic effects appeared within 24 h after transfection,with viral titers stabi-lizing at 107-108 TCID50/mL.Indirect immunofluorescence assay confirmed complete retention of VP1 pro-tein antigenic epitopes and successful expression of recombinant virus capsid protein.Moreover,replication kinetics of the recombinant virus highly resembled the parental strain,with no signifi-cant difference in plaque morphology.EcoR V restriction analysis and sequencing verified the precise introduction and stable inheritance of mutation sites(C7183T and A7186C).In conclusion,the estab-lished DNA-launched system achieved highly efficient FCV rescue and precise genetic inheritance.