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

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 fluorescen...

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Published inViruses Vol. 12; no. 1; p. 50
Main Authors Hinsberger, Aurélie, Graillot, Benoît, Blachère Lopez, Christine, Juliant, Sylvie, Cerutti, Martine, King, Linda A., Possee, Robert D., Gallardo, Franck, Lopez Ferber, Miguel
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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.
AbstractList 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.
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.
Author Graillot, Benoît
Juliant, Sylvie
Lopez Ferber, Miguel
Hinsberger, Aurélie
King, Linda A.
Possee, Robert D.
Blachère Lopez, Christine
Cerutti, Martine
Gallardo, Franck
AuthorAffiliation 4 Department of Biological & Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 0BP, UK; laking@brookes.ac.uk
7 Institute for Advanced Life Science Technology; ITAV USR3505, 1 Place Pierre Potier, 31000 Toulouse, France
3 CNRS UPS3044 Baculovirus et Thérapie, LabEx-53, 30380 Saint Christol lèz Alès, France; sylvie.juliant@cnrs.fr (S.J.); martine.cerutti@cnrs.fr (M.C.)
1 LGEI, IMT Mines Alès, Institut Mines-Télécom et Université de Montpellier Sud de France, 6 Avenue de Clavières, 30100 Alès, France; aurelie.hinsberger@gmail.com (A.H.); graillot.benoit@gmail.com (B.G.); christine.blachere-lopez@mines-ales.fr (C.B.L.)
6 NeoVirTech SAS, 1 Place Pierre Potier, 31000 Toulouse, France
2 INRA, SPE, 400 route des Chappes BP 167, 06903 Sophia-Antipolis CEDEX, France
5 Oxford Expression Technologies Ltd. BioInnovation Hub, Oxford OX3 0BP, UK; r.possee@oetltd.com
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Snippet Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it...
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StartPage 50
SubjectTerms baculovirus infection
Cell culture
Cloning
Deoxyribonucleic acid
DNA
DNA-binding protein
Environmental impact
fluorescence labelling
Gene expression
Gene loci
Genomes
Infections
Labeling
Life cycles
Nucleotide sequence
Occlusion bodies
Plasmids
Proteins
real-time imaging
Viruses
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Title Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHORTM DNA Labeling Technology
URI https://www.proquest.com/docview/2550316759
https://search.proquest.com/docview/2334222884
https://pubmed.ncbi.nlm.nih.gov/PMC7019957
https://doaj.org/article/4241b069683a49759fddf5c4d30bb781
Volume 12
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