Abstract
Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues.
Highlights
IntroductionTheir genome consists of a single circular double-stranded DNA molecule that ranges in size from 80 to 200 kb
The two components of the ANCHOR3TM system were inserted at two different loci in the Autographa calinornica Multiple Nucleopolyhedrovirus (AcMNPV) genome, to avoid putative interferences
The clones obtained were checked by their fluorescence ability, and the insertions were verified by PCR
Summary
Their genome consists of a single circular double-stranded DNA molecule that ranges in size from 80 to 200 kb. Baculovirus transmission is mainly via the oral route. Viruses 2020, 12, 50 that contain identical genomes but differ in their structure and function. In the occlusion body (OB), the virions are embedded in a proteinaceous matrix, where the major component is a virus-encoded protein called polyhedrin or granulin, according to the virus considered. OBs are responsible for the transmission between hosts, via the environment. The Baculoviridae family is presently composed of four genera, Alphabaculovirus, Gammabaculovirus, and Deltabaculovirus, whose OBs contain polyhedrin and embed many virions.
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