Bertin, Bérangère

Bonnin, Delphine

Boutin Fontaine, Marjorie

Galibert, Lionel

Jacob, Aurélien

Lecomte, Christophe

Merten, Otto-Wilhelm

Rivière, Christel

English

OA OpenAgrar repository

    130  LC50 was 1.3x103 and 3.1x103 OBs/ml for the tested 2nd and 4th instars, respectively. The study reports a new baculovirus that might be used as a promising agent for biological control of the jasmine moth.   Poster / Viruses. Wednesday, 16:30. VI-13   A single baculovirus for the production of recombinant Adeno-Associated Virus 8 vectors  Lionel Galibert; Aurélien Jacob; Bérangère Bertin; Marjorie Boutin Fontaine; Delphine Bonnin; Christophe Lecomte; Christel Rivière; Otto-Wilhelm Merten Genethon, 1bis, rue de l’Internationale, 91002 Evry, France Address for correspondence: galibert@genethon.fr  We have developed a single baculovirus, named “Monobac”, for the production of recombinant Adeno-Associated Virus vectors of serotype 8 (rAAV8) using the Sf9 cell/baculovirus system. In an AcMNPV bacmid devoid of the chitinase and cathepsin a genes, the AAV rep2 and cap8 genes have been inserted at the egt locus, while the recombinant AAV was cloned in the Tn7 site. This system was used for the production rAAV8 encoding the human γ-sarcoglycan gene, of clinical interest for the treatment of LGMD2C (γ-sarcoglycanopathy) myopathy disease. Enhanced rAAV8 productivity was observed in the cell culture and was maintained after purification, compared to production system based on the use of 2 baculoviruses. The produced rAAV8 capsids displayed a reduced degradation profile of the capsid proteins VP1/VP2 due to the elimination of the baculovirus cathepsin protease gene. This optimized system allows the production of an improved quantity of rAAV vectors with improved vector quality, resulting in enhanced infectivity of the rAAV. .    Poster / Viruses. Wednesday, 16:30. VI-14 Determining the role of P10 during baculovirus infection through the development of novel mutants in Autographa californica multicapsid nucleopolyhedrovirus Leo Graves1, Farheen Raza1, Sarah L. Irons1; Robert D Possee1,2 & Linda A King1 1Department of Biological and Medical Sciences, Oxford Brookes University, Oxford UK, 2Oxford Expression Technologies Ltd, Oxford, UK. Address for Correspondence: 12083983@brookes.ac.uk  P10 is a fibrous protein that forms complex networks of filaments and a distinct perinuclear tubular structure around the nucleus during the later stages of infection of cells with baculovirus.  Previous research has suggested possible roles of P10 in nuclear stability, polyhedron formation and cell lysis, but distinct functional roles for the protein have yet to be determined. In order to investigate the role of P10 during infection, a variety of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) mutants have been constructed, which include a p10 deletion and associated rescue virus, phosphorylation mutants and a virus in which the AcMNPV p10 coding region has been replaced with that from Spodoptera frugiperda (Sf)NPV. Mass spectrometry was used to confirm the phosphorylation of P10 serine 93.  Mutation of serine 93 to alanine affected the structure of P10 tubules as evidenced by confocal microscopy.  The distinctive tubular structure surrounding the nucleus that is observed in wild-type virus infected cells failed to form correctly.  Circular dichroism analysis confirmed a distinct change in the protein secondary structure.  These data suggest that phosphorylation plays a key role in P10 function. Replacement of the AcMNPV p10 coding region with that from SfNPV resulted in a virus with low budded virus titre and aberrant rearrangement of microtubules in comparison to AcMNPV-infected cells, suggesting that the SfNPV P10 may be affecting microtubules and translocation of nucleocapsids to the plasma membrane for budding.     Poster / Viruses. Wednesday, 16:30. VI-15   Evaluation of the transcriptional transactivation of betabaculovirus regulatory elements in transformed cell lines by alphabaculovirus transcription factors Santiago Haase1, M. Leticia Ferrelli1; Matías L. Pidre1; Alicia Sciocco-Cap2, Víctor Romanowski1 1IBBM-UNLP-CONICET, La Plata, AR; 2IMyZA-INTA, Castelar, AR; Address for Correspondence: santiagohaase@biol.unlp.edu.ar  The narrow host range of baculoviruses is one of their advantages for sustainable pest control of a single insect species with minimal or no effect on non-target organisms. However, the limited host range may be less desirable from the economical point of view since more than one pest can be present at the same time on most crops. Engineering baculovirus genomes with an expanded host range by design would be an answer to this type of scenarios. Nevertheless, genetic determinants of host range have not been widely characterized and the mechanisms of host recognition are still not well understood. In this context, the generation of hybrid or chimaeric baculoviruses may be an empirical approach to generate viruses with expanded host range. The expression of viral genes in this context will require the transcriptional transactivation of their promoters by heterologous transcription factors (TFs). However, the recognition of baculovirus promoters in different species has not been systematically studied so far. The aim of our work is to evaluate the transcriptional transactivation of betabaculovirus promoters by alphabaculovirus TFs. It has been noted before that late promoters require the replication of the DNA to be activated. Therefore, we generated stably transformed cell lines expressing the red fluorescent protein (DsRed) as a reporter gene under the control of immediate-early, early and late gene promoters of the Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) and Epinotia aporema granulovirus (EpapGV), respectively. These cell lines were infected with AgMNPV to evaluate the transcriptional transactivation of these promoters. Our results showed that the AgMNPV transcription factors activate early and late EpapGV promoters.     Poster / Viruses. Wednesday, 16:30. VI-16   Enhancin Genes of Lymantria dispar NPV Do Not Increase Potency Via Metalloprotease Activity Kelli Hoover1, James Slavicek2, Algimantas P. Valaitis2,3, Nancy Hayes-Plazolles2, and Elizabeth McCarthy1 1 Department of Entomology, Penn State University, University Park, PA USA, 2 USDA Forest Service, Delaware, OH USA, 3Retired Address for Correspondence: kxh25@psu.edu  The baculovirus encoded enhancins characterized so far are metalloproteases that digest proteins in the peritrophic matrix (PM) of their host midgut, increasing viral potency in many systems. Lymantria dispar NPV has two enhancin genes (E1 and E2) that are distributed in the ODV envelopes, placing them in a position to interact with the PM and the and possibly midgut cells. Deletion of either enhancin or both reduces viral potency 2-fold and 12-fold, respectively, compared to wildtype. Removal of the PM with optical brightener treatment did not alter these differences in potencies, suggesting that the enhancins do not affect the PM. The results of an in vitro PM digestion assay found that although the PM was degraded, it was not affected by inhibitors of metalloproteases, whereas treatment with serine protease inhibitors showed little or no PM degradation. Mutant LdNPV viruses were generated by altering the region that encodes the zinc binding site of the metalloprotease; this region of E1 and E2 was either deleted or altered by homologous amino  

A single baculovirus for the production of recombinant Adeno-Associated Virus 8 vectors

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A single baculovirus for the production of recombinant Adeno-Associated Virus 8 vectors

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