Generation of a transposon insertion mutant library for bovine herpesvirus 4 cloned as a bacterial artificial chromosome by in vitro MuA based DNA transposition system

2007 ◽  
Vol 141 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Gaetano Donofrio ◽  
Eugenio Martignani ◽  
Chiara Sartori ◽  
Alain Vanderplasschen ◽  
Sandro Cavirani ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Bovine Herpesvirus ◽  
Artificial Chromosome ◽  
Insertion Mutant ◽  
Mutant Library ◽  
Dna Transposition ◽  
Transposon Insertion ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 4

scholarly journals Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning

2005 ◽  
Vol 86 (4) ◽  
pp. 907-917 ◽  
Author(s):  
L. Gillet ◽  
V. Daix ◽  
G. Donofrio ◽  
M. Wagner ◽  
U. H. Koszinowski ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Expression Vector ◽  
Bovine Herpesvirus ◽  
Artificial Chromosome ◽  
Complement Protein ◽  
Bovine Herpesvirus 4 ◽  
Infected Cells ◽  
Bac Cloning ◽  
Herpesvirus 4 ◽  
Bacterial Artificial Chromosome Cloning

Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.

scholarly journals Molecular Characterization of the First Bovine Herpesvirus 4 (BoHV-4) Strains Isolated from In Vitro Bovine Embryos production in Argentina

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0132212 ◽  
Author(s):  
Erika González Altamiranda ◽  
Julieta M. Manrique ◽  
Sandra E. Pérez ◽  
Glenda L. Ríos ◽  
Anselmo C. Odeón ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Bovine Herpesvirus ◽  
Bovine Embryos ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 4

scholarly journals Cellular Targeting of Engineered Heterologous Antigens Is a Determinant Factor for Bovine Herpesvirus 4-Based Vaccine Vector Development

2009 ◽  
Vol 16 (11) ◽  
pp. 1675-1686 ◽  
Author(s):  
Gaetano Donofrio ◽  
Valentina Franceschi ◽  
Antonio Capocefalo ◽  
Simone Taddei ◽  
Chiara Sartori ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
Bovine Herpesvirus ◽  
Artificial Chromosome ◽  
Diarrhea Virus ◽  
Chimeric Peptide ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 4 ◽  
Protein Cell

ABSTRACT In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.

Bovine herpesvirus 4 based vector interaction with liver cells in vitro and in vivo

2006 ◽  
Vol 136 (1-2) ◽  
pp. 126-136 ◽  
Author(s):  
Gaetano Donofrio ◽  
Eugenio Martignani ◽  
Enzo Poli ◽  
Claudia Lange ◽  
Filippo Maria Martini ◽  
...  
Keyword(s):  
Bovine Herpesvirus ◽  
Liver Cells ◽  
Vector Interaction ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 4

scholarly journals Bovine herpesvirus 4 ORF73 is dispensable for virus growth in vitro, but is essential for virus persistence in vivo

2010 ◽  
Vol 91 (10) ◽  
pp. 2574-2584 ◽  
Author(s):  
M. Thirion ◽  
B. Machiels ◽  
F. Farnir ◽  
G. Donofrio ◽  
L. Gillet ◽  
...  
Keyword(s):  
Bovine Herpesvirus ◽  
Virus Persistence ◽  
Virus Growth ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 4

scholarly journals The core 2 β-1,6-N-acetylglucosaminyltransferase-M encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins

2004 ◽  
Vol 85 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Nicolas Markine-Goriaynoff ◽  
Laurent Gillet ◽  
Odd A. Karlsen ◽  
Lars Haarr ◽  
Frédéric Minner ◽  
...  
Keyword(s):  
Virus Replication ◽  
Bovine Herpesvirus ◽  
Early Gene ◽  
Structural Proteins ◽  
Phylogenetic Study ◽  
African Buffalo ◽  
Post Translational Modifications ◽  
Bovine Herpesvirus 4 ◽  
Herpesvirus 4

The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only virus gene known to date that encodes a homologue of the cellular core 2 β-1,6-N-acetylglucosaminyltransferase-mucine type (C2GnT-M). Recently, our phylogenetic study revealed that the Bo17 gene has been acquired from an ancestor of the African buffalo around 1·5 million years ago. Despite this recent origin, the Bo17 sequence has spread to fixation in the virus population possibly by natural selection. Supporting the latter hypothesis, it has been shown by our group for the V. test strain that Bo17 is expressed during BoHV-4 replication in vitro, and that Bo17 expression product (pBo17) has all three enzymic activities exhibited by cellular C2GnT-M, i.e. core 2, core 4 and I branching activities. In the present study, firstly it was investigated whether encoding a functional C2GnT-M is a general property of BoHV-4 strains. Analysis of nine representative strains of the BoHV-4 species revealed that all of them express the Bo17 gene and the associated core 2 branching activity during virus replication in vitro. Secondly, in order to investigate the roles of Bo17, its kinetic class of expression was analysed and a deleted recombinant strain was produced. These experiments revealed that Bo17 is expressed as an early gene which is not essential for virus replication in vitro. However, comparison of the structural proteins, produced by the wild-type, the revertant and the deleted viruses, by 2D gels demonstrated that pBo17 contributes to the post-translational modifications of structural proteins. Possible roles of Bo17 in vivo are discussed.

scholarly journals A bovine macrophage cell line supports bovine herpesvirus-4 persistent infection

2001 ◽  
Vol 82 (5) ◽  
pp. 1181-1185 ◽  
Author(s):  
Gaetano Donofrio ◽  
Vicky L. van Santen
Keyword(s):  
Viral Genome ◽  
Bovine Herpesvirus ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Persistently Infected ◽  
Bovine Herpesvirus 4 ◽  
Bovine Macrophage ◽  
Herpesvirus 4

Although bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, has been demonstrated in many tissues during persistent BHV-4 infection, a likely site of virus persistence is in cells of the monocyte/macrophage lineage. To establish an in vitro model of persistent infection potentially useful for examining the molecular mechanisms of BHV-4 persistence/latency, we infected the bovine macrophage cell line BOMAC. Following extensive cell death, surviving cells were found to be persistently infected, maintaining the viral genome over many passages and producing low levels of infectious virus. Although selection was unnecessary for the maintenance of the viral genome, cells persistently infected with recombinant BHV-4 containing a neomycin-resistance gene could be selected with geneticin, thus confirming that persistent BHV-4 infection was compatible with cell survival and replication. Furthermore, persistent BHV-4 infection caused no decrease in the growth rate of BOMAC cells. Sodium butyrate, which reactivates latent gammaherpesviruses in vitro, or dexamethasone, which reactivates latent BHV-4 in vivo, increased viral DNA by 10- to 15-fold in persistently infected BOMAC cells. This suggests that reactivation of latent BHV-4 by dexamethasone in vivo might involve direct action of dexamethasone on latently infected cells.

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