Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHORTM DNA Labeling Technology

Viruses

卷:12

RobertD. Possee¹  Sylvie Juliant²  Martine Cerutti²  LindaA. King³  Aurélie Hinsberger⁴  Christine Blachère Lopez⁴  Benoît Graillot⁴  Miguel Lopez Ferber⁵  Franck Gallardo⁶ 

[1] Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, UK;

[2] CNRS UPS3044 Baculovirus et Thérapie, LabEx-53, 30380 Saint Christol lèz Alès, France;

[3] Department of Biological &

[4] LGEI, IMT Mines Alès, Institut Mines-Télécom et Université de Montpellier Sud de France, 6 Avenue de Clavières, 30100 Alès, France;

[5] NeoVirTech SAS, 1 Place Pierre Potier, 31000 Toulouse, France;

[6] Oxford Expression Technologies Ltd. BioInnovation Hub, Oxford OX3 0BP, UK;

关键词: real-time imaging;    fluorescence labelling;    baculovirus infection;   

DOI  :  10.3390/v12010050

来源: DOAJ

【 摘 要 】

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 ANCHOR^TM 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 ANCHOR^TM: 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.

【 授权许可】

Unknown