A nucleocapsid-tagged fluorescent pangolin coronavirus enables authentic-virus neutralization assays and localizes the antiviral activity of cepharanthine to viral entry

Replication-competent coronaviruses carrying a fluorescently tagged structural protein are rare, and the only well-developed example tags the membrane (M) protein. Using the highly attenuated pangolin coronavirus GX_P2V(short_3UTR) as a backbone, we assembled a full-length cDNA clone by multifragment in vitro ligation and recovered GX_P2V-mBJ-N, a recombinant virus in which the bright monomeric green fluorescent protein mBaoJin is fused to the N terminus of the nucleocapsid (N) protein. The equivalent C-terminal fusion was not viable. GX_P2V-mBJ-N is infectious in Vero E6 cells but is attenuated and genetically unstable in the absence of exogenous N; supplying wild-type N in trans allows propagation to high titer with the fusion intact. Because mBaoJin is translated as part of N, fluorescence reports viral antigen synthesis directly, and infection can be quantified in a plate reader without immunostaining or plaque reduction. We applied the reporter virus in two settings. First, sera from ten healthy blood donors were titrated in parallel against GX_P2V-mBJ-N and a vesicular stomatitis virus (VSV)-based GX_P2V pseudovirus. The two assays agreed on rank order, but pseudovirus titers were about 10-fold higher overall and diverged by at least 70-fold in one donor, showing that the two formats are not interchangeable for individual samples. Second, time-of-addition experiments identified entry as the principal target of cepharanthine (EC50, 0.20 M under continuous treatment), whereas preincubation of virions with 25 M drug had no effect, excluding direct virion inactivation. GX_P2V-mBJ-N provides a practical authentic-virus platform for coronavirus serology and antiviral pharmacology.