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Updated: Mar 31, 2026
Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus
Published on: August 23, 2008
Bombyx mori nucleopolyhedrovirus (BmNPV) Bm64 is required for BV production and per os infection
Lin Chen1,2, Yunwang Shen2, Rui Yang2
1Sericultural Research Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.
Background:
Bombyx mori nucleopolyhedrovirus (BmNPV) orf64 (Bm64, a homologue of ac78) is a core baculovirus gene. Recently, Li et al. reported that Ac78 was not essential for budded viruses (BVs) production and occlusion-derived viruses (ODVs) formation (Virus Res 191:70-82, 2014). Conversely, Tao et al. demonstrated that Ac78 was localized to the BV and ODV envelopes and was required for BV production and ODV formation (J Virol 87:8441-50, 2013). In this study, the function of Bm64 was characterized to determine the role of Bm64 in the BmNPV infection cycle.
Method:
The temporal expression of Bm64 was examined using total RNA extracted from BmNPV-infected BmN cells at different time points by reverse-transcription PCR (RT-PCR) and 5' RACE analysis. To determine the functions of Bm64 in viral replication and the viral phenotype throughout the viral life cycle, a deletion virus (vBm(64KO)) was generated via homologous recombination in Escherichia coli. Viral replication and BV production were determined by real-time PCR. Electron microscopy was used to detect virion morphogenesis. The subcellular localization of Bm64 was determined by microscopy, and per os infectivity was used to determine its role in the baculovirus oral infection cycle.
Results:
Viral plaque and titer assay results showed that a few infectious BVs were produced by vBm(64KO), suggesting that deletion of Bm64 affected BV production. Viral DNA replication was detected and polyhedra were observed in vBm(64KO)-transfected cells. Microscopy analysis revealed that Bm64 was predominantly localized to the ring zone of the nuclei during the infection cycle. Electron microscopy showed that Bm64 was not essential for the formation of ODVs or the subsequent occlusion of ODV into polyhedra. The per os infectivity results showed that the polyhedra of vBm(64KO) were unable to infect silkworm larvae.
Conclusion:
In conclusion, our results suggest that Bm64 plays an important role in BV production and per os infection, but is not required for viral DNA replication or ODV maturation.
Insights
Bombyx mori nucleopolyhedrovirus orf64 (Bm64) is crucial for budded virus (BV) production and oral infection in silkworms. However, Bm64 is not essential for viral DNA replication or occlusion-derived virus (ODV) formation.
Area of Science:
- Virology
- Molecular Biology
- Insect Pathology
Background:
- Bombyx mori nucleopolyhedrovirus (BmNPV) orf64 (Bm64) is a core baculovirus gene with debated roles in virus production.
- Conflicting reports exist regarding the necessity of its homologue, Ac78, for budded virus (BV) and occlusion-derived virus (ODV) formation.
Purpose of the Study:
- To elucidate the function of Bm64 in the BmNPV infection cycle.
- To determine the role of Bm64 in viral replication, virion production, and oral infectivity.
Main Methods:
- Temporal expression analysis using RT-PCR and 5' RACE.
- Generation of a Bm64 deletion virus (vBm(64KO)) via homologous recombination.
- Assessment of viral replication, BV production, virion morphogenesis, subcellular localization, and per os infectivity.
Main Results:
- Deletion of Bm64 significantly reduced infectious BV production.
- Viral DNA replication and polyhedra formation were observed in vBm(64KO)-infected cells.
- Bm64 localizes to the nucleus; it is not essential for ODV formation or occlusion, but vBm(64KO) polyhedra lost oral infectivity.
Conclusions:
- Bm64 is essential for efficient BV production and per os infectivity of BmNPV.
- Bm64 is dispensable for viral DNA replication and ODV maturation.
- The findings clarify the specific roles of Bm64 in the baculovirus life cycle.
