Construction and characterization of a recombinant invertebrate iridovirus

Özgen, Arzu; Muratoglu, Hacer; Demirbağ, Zihni; Vlak, Just M.; Oers, Monique M. van; Nalçacıoğlu, Remziye

doi:10.1016/j.virusres.2014.05.012

Cited by 11 publications

(8 citation statements)

References 60 publications

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“…While transcriptomic and proteomic studies may address some of these short-comings, other important questions regarding iridovirid-host interactions cannot be addressed without the use of functional genomics. Previously, functional genomics studies in IIVs have been hampered by the lack of genetic recombination systems for producing genetic (knockout) mutants of the virus; however, the first recombinant IIV using a homologous recombination site was recently established [ 161 , 162 ]. The identification of viral proteins responsible for viral infectivity will be an important next step towards the development of a genetic recombination system for iridovirids (possibly similar to a bacmid system) as iridovirid DNA by itself is not infectious.…”

Section: Discussionmentioning

confidence: 99%

Invertebrate Iridoviruses: A Glance over the Last Decade

İnce

Özcan

Ilter-Akulke

et al. 2018

Viruses

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Members of the family Iridoviridae (iridovirids) are large dsDNA viruses that infect both invertebrate and vertebrate ectotherms and whose symptoms range in severity from minor reductions in host fitness to systemic disease and large-scale mortality. Several characteristics have been useful for classifying iridoviruses; however, novel strains are continuously being discovered and, in many cases, reliable classification has been challenging. Further impeding classification, invertebrate iridoviruses (IIVs) can occasionally infect vertebrates; thus, host range is often not a useful criterion for classification. In this review, we discuss the current classification of iridovirids, focusing on genomic and structural features that distinguish vertebrate and invertebrate iridovirids and viral factors linked to host interactions in IIV6 (Invertebrate iridescent virus 6). In addition, we show for the first time how complete genome sequences of viral isolates can be leveraged to improve classification of new iridovirid isolates and resolve ambiguous relations. Improved classification of the iridoviruses may facilitate the identification of genus-specific virulence factors linked with diverse host phenotypes and host interactions.

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Section: Discussionmentioning

confidence: 99%

Invertebrate Iridoviruses: A Glance over the Last Decade

İnce

Özcan

Ilter-Akulke

et al. 2018

Viruses

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“…While lamellocyte differentiation is generally induced during parasitic wasp infection 25 , wounding of the Drosophila larvae is also sufficient to induce lamellocyte differentiation 26 . Finally, while not utilized in the experiments presented here, there are GFP-expressing forms of Listeria 27 and IIV6 28 that could be used to generate 3D models of hemocyte infection. Instead, Listeria - and IIV6-infected hemocytes were used for Western blotting and qRT-PCR experiments.…”

Section: Representative Resultsmentioning

confidence: 99%

Extraction of Hemocytes from <em>Drosophila melanogaster</em> Larvae for Microbial Infection and Analysis

Hiroyasu

DeWitt

Goodman

2018

JoVE

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During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 × 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

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“…FV3-vIF-2␣, FV3-18K, 64R-FV3, 52L-FV3 and ESV DHFR were generated basing on fluorescence marker coupled with resistance gene (Chen et al, 2011;Andino et al, 2015;Martín et al, 2015). The other six, including 67R-RGV, TK-RGV, i53R-RGV-lacIO, i2L-RGV-lacIO, EGFP-STIV and rCIV-157L-gfp, were purified by successive rounds of plaque isolation using fluorescence selection (He et al, 2012;Huang et al, 2014;Ozgen et al, 2014;He et al, 2013;Huang et al, 2011;He et al, 2014). The successfully purification of DUT, TK-RGV suggested that fluorescence alone was competent for recombinant virus construction, which was also confirmed by the generation of several recombinant vaccinia virus and herpesvirus (Maruri-Avidal et al, 2011;Fuchs et al, 2011;Satheshkumar et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…were completely knocked out (Jancovich and Jacobs, 2011;Ozgen et al, 2014;He et al, 2014). Moreover, we would have two loci for immune gene expression, which may enlighten the development of genetically modified live vaccines (Paillister et al, 2007).…”

Section: Introductionmentioning

confidence: 99%