Location: Office of The Director
Project Number: 5090-31320-004-02-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2016
End Date: Jun 30, 2019
To address the needs of regional salmonid aquaculture, this agreement seeks to build upon previous work characterizing the mechanisms of virulence and immune evation utilized by viral hemorrhagic septicemia virus (VHSV), as well as expansion of these studies to include characterization of infectious hematopoietic necrosis virus (IHNV). Specifically, this work will focus on identification of critical portions of viral proteins involved in suppression of host cell gene expression and the control of host cell death. To do so, the following objectives have been identified: 1) Compile a sequence database of M protein diversity to identify residues with potential roles in pathogensis using structural modeling. 2) Generate and assess M protein mutants using in vitro transcriptional repression assays. 3) Compile a sequence database of NV protein diversity to identify residues with potential roles in pathogenesis. 4) Generate and assess NV protein mutants using in vitro apoptosis assays 5) Modify existing IHNV infectious clone to facilitate transfer of gene cassettes containing mutations of interest.
1) Environmental viral hemorrhagic septicemia virus (VHSV) isolates will be sequenced and analyzed bioinformatically, along with previously obtained and publicly available sequences, using CLUSTAL_X. Available infectious hematopoietic necrosis virus (IHNV) sequences will be assessed in parallel. Regions of the M gene that will be characterized by degree of sequence variability, and structural modeling will be performed to predict regions that are surface exposed and likely to be involved in protein interactions. These regions will be targeted for mutagensis, as will other sites inferred from isolates known to exhibit enhanced cytopathicity. Wherever possible, these sites will be chosen using cell-based studies in which cellular cytopathicity will be used as a surrogate of virulence. 2) At least four M protein expression constructs will be generated harboring unique mutations in the background of the IHNV(220-90) and VHSV(IVb) M genes. Each of the mutant proteins will be assessed for transcriptional repression in EPC and RTG-2 cell lines using both luciferase reporter assays as well as assessment of nascent transcription rates in transfected cells using EU/Click-iT labeling. All clones will also be assessed for apoptotic activity in tranfected cells, using assays described below (4). 3) NV sequences from the same viral genomes sequenced above (1) will be similarly analyzed to identify site to target for mutagenesis. This analysis will be coupled with any available virulence data to determine whether observable differences correlate with viral pathogenicity. In the absence of such data, amino acid changes that would be predicted to alter protein structure, pI or charge distribution will be incorporated into expression plasmids for use in the cell-based the apoptosis studies described below. 4) At least four NV protein expression constructs will be generated harboring unique mutations in the background of the IHNV(220-90) and VHSV(IVb) NV genes. Each of the mutant proteins will be assessed for induction/repression of apoptosis in both EPC and RTG-2 cell lines. Multiple apoptosis assays in EPC cells using a wide array of inducers have already been standardized; however, these assays have not been validated and compared in RTG-2 cells. Established assays will be similarly attempted using RTG-2 cells, and assays which exhibit the greatest degree of reproducibility, as well as continuity with results obtained using EPC cells, will be standardized for use in the assessment of viral protein mutants. 5) A reverse genetic system (infectious clone) for IHNV (220-90) has been developed, however performing genetic manipulation is cumbersome in the context of the current clone. To better facilitate transfer of mutated genes, unique restriction enzyme recognition sequences will be engineered into the full-length clone, flanking the NV and M genes. Recombinant viral stocks will be generated and viral titer obtained by plaque assay on EPC and RTG-2 cells. Replication of the modified recombinant virus will be compared to the parental virus, to ensure that the introduced changes have not had a detrimental effect on viral growth.