Development of a DNA plasmid-based approach for efficient synthesis of Sacbrood virus infectious clones within host cells

Authors: YUE, DANDAN - Fujian Agricultural & Forestry University; LI, RUNLIN - Fujian Agricultural & Forestry University; ZHANG, KAILANG - Fujian Agricultural & Forestry University; Chen, Yanping - Judy; Palmer-Young, Evan; HUANG, SHAOKANG - Fujian Agricultural & Forestry University; Huang, Wei

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2023
Publication Date: 8/18/2023
Citation: Yue, D., Li, R., Zhang, K., Chen, Y., Palmer-Young, E.C., Huang, S., Huang, W. 2023. Development of a DNA plasmid-based approach for efficient synthesis of Sacbrood virus infectious clones within host cells. Viruses. https://doi.org/10.3390/v15091866.
DOI: https://doi.org/10.3390/v15091866

Interpretive Summary: The lack of an effective cell culture system for virus propagation has hampered research on the replication and disease mechanisms of honeybee viruses. In the present study, we employed an insect cell-insect baculovirus system for the generation of the honeybee Sacbrood virus (SBV). Our results clearly showed that the synthesis of bee-infecting RNA viruses using the insect cell-insect baculovirus system is feasible and suggest that the insect cell-insect baculovirus system is a powerful platform for studies of fundamental biological processes of honeybee viruses. The results obtained from our study should be of interest to researchers, graduate students, regulators, and beekeepers worldwide.

Technical Abstract: The decline of both domestic honey bees and wild pollinators is frequently attributed to RNA viruses. To expedite the development of effective countermeasures against these viruses, a more comprehensive understanding of virus biology necessitates extensive collaboration among scientists from diverse research fields. While the infectious virus clone is a robust tool for studying virus diseases, the current methods for synthesizing infectious clones of bee-infecting RNA viruses entail in vitro transcription of the viral genome RNA in 8-10kb, presenting challenges in reproducibility and distribution. This article reports on the synthesis of an infectious clone of Sacbrood virus (SBV) using a DNA plasmid containing an Autographa californica nucleopolyhedrovirus (AcMNPV) immediate-early protein (IE1) promoter to trigger transcription of the downstream viral genome within hosts. The results demonstrate that the IE1-SBV plasmid can synthesize SBV clones in a commonly used lepidopteran immortal cell line (Sf9) and honey bee pupae. Moreover, the negative strand of the clone was detected within Sf9, indicating active infection and replication. However, the synthesized clone did not seem to disseminate to adjacent cells, and only approximately 10% of cells were transfected by the plasmid. Injection of honey bee pupae with the IE1-SBV plasmid resulted in a low infection rate of 20-30%, comparable to the results of our previous experiments involving the injection of pupae with in vitro transcribed viral RNA of the same clone. Our results suggest that the synthesis of bee-infecting RNA viruses using DNA plasmids is feasible, facilitating streamlined production of clones with various sequence editing to reveal viral gene functions and biology. The ease of distributing infectious clones in DNA plasmid status may increase collaboration among scientists in applying the clone to bee biology, ecology, and behavior, ultimately providing a comprehensive approach to managing virus diseases in the future.