Spatially Segregated Transmission of Co-Occluded Baculoviruses Limits Virus-Virus Interactions Mediated by Cellular Coinfection during Primary Infection.

The occlusion bodies (OBs) of certain alphabaculoviruses are polyhedrin-rich structures that mediate the collective transmission of tens of viral particles to the same insect host. In addition, in multiple nucleopolyhedroviruses, occlusion-derived virions (ODVs) form nucleocapsid aggregates that are delivered to the same host cell. It has been suggested that, by favoring coinfection, this transmission mode promotes evolutionarily stable interactions between different baculovirus variants. To quantify the joint transmission of different variants, we obtained OBs from cells coinfected with two viral constructs, each encoding a different fluorescent reporter, and used them for inoculating Spodoptera exigua larvae. The microscopy analysis of midguts revealed that the two reporter genes were typically segregated into different infection foci, suggesting that ODVs show limited ability to promote the co-transmission of different virus variants to the same host cell. However, a polyhedrin-deficient mutant underwent inter-host transmission by exploiting the OBs of a fully functional virus and re-acquired the lost gene through recombination, demonstrating cellular coinfection. Our results suggest that viral spatial segregation during transmission and primary infection limits interactions between different baculovirus variants, but that these interactions still occur within the cells of infected insects later in infection.

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing.

The clustered regularly interspaced short palindromic repeats (CRISPR) system-associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.