Construction of infectious clone of raccoon dog and arctic fox amdoparvovirus and virus rescue
The aim was to establish a reverse genetic system for raccoon dog and arctic fox amdoparvovirus(RFAV)and to provide a platform for studying the pathogenic mechanisms of amdoparvo-viruses and vaccine development at the whole gene level.Our laboratory has successfully obtained the sequence information of RFAV middle coding region(M1,M2,M3)and terminal sequences through segmental cloning.The sequences of the middle coding regions M1,M2,M3,and the synthetic terminal coding region sequences of RFAVwere constructed into the expression vector pBBSma Ⅰ using seamless cloning technology.The full-length plasmid pBB-RFAV was obtained,and this plasmid was then transfected into F81 cells for virus rescue.Verification of the rescued virus was conducted through observations of cytopathic effects,PCR detection,qPCR detection of the relative expression of the virus,indirect immunofluo-rescence assay(IFA)of rRFAV,and virion electron microscopy.Results indicated that signifcant cyto-pathic changes were observed in the cells at 48 hours,and RFAV fragments were detected by PCR.qPCR confirmed that the rescued virus could be stably passaged in F81 cells,exhibiting proliferation dynamics similar to that of Aleutian mink disease virus(AMDV).IFA results demonstrated specific fluo-rescent signals in infected cells,and the particle morphology of the rescued virus was consistent with the known morphological characteristics of AMDV according to electron microscopy.These findings highlight the successful construction of the whole-genome infectious clone of RFAV,leading to the successful rescue of the virus rRFAV.This achievement lays the foundation for further studies on the etiology and immunology of amdoparvoviruses.
raccoon dog and arctic fox amdoparvovirusinfectious clonevirus rescuereverse ge-netic system
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