Abstract: 【Objective】A vp39 pseudotyped Autographa californica multiple nucleopolyhedrovirus(AcMNPV) in which AcMNPV vp39 was replaced with Spodoptera exigua MNPV(SeMNPV) VP39 was constructed to determine whether SeMNPV VP39 could replace AcMNPV VP39 in AcMNPV. This study laid a theoretical foundation for further exploring the nucleocapsid assembly mechanism of baculovirus.【Method】Via Tn7-mediated transposition, the SeMNPV vp39 gene under the control of the AcMNPV vp39 promoter with a FLAG tag prior to the SeMNPV vp39 stop codon, together with the enhanced green fluorescence protein(egfp) and polyhedrin(polh) genes, was inserted into the AcMNPV vp39 knockout bacmid(bAcvp39 KO) polh locus to construct vp39 pseudotyped AcMNPV(vAcSevp39:FLAG). Western blotting was performed to determine whether SeMNPV vp39 was expressed in Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9(Sf9) cells transfected with vAcSevp39:FLAG. The viral replication and infection in Sf9 cells transfected with vAcSevp39:FLAG were monitored using a fluorescence microscope. Viral growth curve analysis was performed by 50%tissue culture infective dose(TCID₅₀) endpoint dilution assay to further evaluate the ability of vAcSevp39:FLAG to produce infectious budded virion(BV). Thin section of vAcSevp39:FLAG-transfected Sf9 cells was observed using an electron microscope to investigate the virus morphogenesis.【Result】Western blotting showed that SeMNPV VP39 was detected in Sf9 cells transfected with vAcSevp39:FLAG. Fluorescence microscopy and viral growth curve analysis showed that no infectious BVs were produced in vAcSevp39:FLAG-transfected Sf9 cells, which was consistent to AcMNPV vp39 knockout recombinant virus(vAcvp39 KO). Electron microscopy demonstrated that, different from vAcvp39 KO, in Sf9 cells transfected with vAcSevp39:FLAG, masses of abnormally elongated empty capsid structures were observed inside the nuclei, but no nucleocapsids formed.【Conclusion】SeMNPV VP39 has the ability to assemble tubular capsid-like structures but not nucleocapsids in AcMNPV, resulting in no BV and occlusion-derived virion produced.