Generation And Characterization Of The AcMNPV-Bombyx Mori Bidensovirus Chimeras

Bombyx mori bidensovirus(BmBDV),belongs to the family of Bidnaviridae and the genus of Bidensovirinea.BmBDV contains two sets of complementary linear single-stranded DNA molecules about 6.5 kb(VD1)and 6 kb(VD2).BmBDV mainly replicates in the midgut columnar epithelial cells of the silkworm,causing the disease similar to insect densonucleosis.It is one of the main pathogens for the economic reduction of the silkworm industry.At present,there is no cell line in vitro to support the replication of BmBDV,which greatly limits the research on the proliferation and gene function of BmBDV.The mechanism of BmBDV invasion and replication,the gene function of BmBDV,the regulation mechanism of expression of viral genes and so on are far from clear.It is very important to build a stable and efficient reverse genetic system that can rescue BmBDV.It is an effective strategy to rescue the viruses from cloning plasmids.Although some scholars expect to construct the recombinant plasmids containing BmBDV genome to achieve the rescue of BmBDV,there are still problems such as lowefficiency of rescue,and no specific markers.The genome of AcMNPV can be regarded as a large plasmid,which can contain about 100 kb of exogenous DNA and replicate efficiently in sensitive cells.In this study,we use Bac-to-Bac system to insert the genome of BmBDV into AcMNPV,construct AcMNPV and BmBDV chimeric recombinant virus,and analyze the replication characteristics of chimeric virus.First,we fused Flag tags at the C-terminal of NS1 and VP respectively without destroying the integrity of the VD1 genome,inserted complete eGFP expression box into the noncoding region of VD2 genome,and successfully constructed the recombinant plasmids including BmBDV genome with specific markers: pVD1-NS1 Flag,pVD1-VPFlag and pVD2-GFP.The expression of NS1-Flag and VP-Flag proteins as well as the green fluorescence can be detected in Hi5 cells transfected by recombinant plasmid.Then,using Bac-to-Bac system,the chimeric viruses Ac-VD1/NS1 Flag,Ac-VD1/VPFlag and Ac-VD2 GFP containing VD1 or VD2 with specific tags were successfully constructed.The results of RT-PCR and Western Blot showed that BmBDV genes can be transcribed and expressed along with the replication of chimeric viruses in insect Hi5 cells,and the expression pattern was similar to that of exprssion in midgut of BmBDV-infected silkworm.Then,Hi5 cells was infected with chimeric virus alone or co-infect with Ac-VD1 and Ac-VD2,cell lysate was collected at 96 hours after infection and fed to the second instar larvae of the susceptible strain Huabasanwu.No typical symptoms of BmBDV infection were observed in the silkworm fed with the lysate of the cells infected by chimera virus,and no expression of eGFP was observed in the midgut of the silkworms.The expression of BmBDV gene were not detected by RT-PCR and western blot analysis.These results showed that BmBDV was not rescued along with the replication of the recombinant virus in Hi5 cells.The results of Southern blot showed that the BmBDV genome could not be rescued from chimeric virus in Hi5 cells infected by recombinant virus.In summary,we have successfully constructed the chimeric BmBDV-AcMNPV with specific markers.The genes of BmBDV can be expressed along with the replication of chimeric viruses in Hi5 cells,but the infectious BmBDV particles cannot be rescued,because the genome of BmBDV cannot be released from the genome of chimeric virus.Our research provides a convenient platform for further study of the expression and regulation mechanism of BmBDV genes,and lays a foundation for the creation of new biological control agents,such as chimeric virus of baculovirus,densonucleosis virus and other insect viruses.