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Principal Investigator: Nikolaus Osterrieder
Contact Information: Email: no34@cornell.edu - Phone: 607-253-4045
Sponsor: Grayson-Jockey Club Research Foundation, Inc.
Grant Number: N/A
Title: The Neurologic EHV-1 Marker: Correlation or Causation?
Annual Direct Cost: $47,800
Project Period: 06/01/07-05 /31/08
DESCRIPTION (provided by applicant): The hypothesis to be tested in this proposal is that a point mutation in equine herpesvirus type 1 (EHV 1) ORF30, which encodes the catalytic subunit of the DNA polymerase, induces an altered cellular tropism for horse peripheral blood mononuclear cells (PBMC). The hypothesis is based on the observation that a particular genotype encoding a aspartic acid residue at position 752 of the enzyme (D752) is predominantly found in neuropathogenic ("neurologic") EHV-1 strains, while an asparagines residue (N752) predominates in non-neurologic ("abortion-only") strains [31]. Since the only animal model for the neurologic outsome of this disease is the horse, and neurologic challenge trials are costly and require difficult subjective assessments, we propose a number of in vitro experiments to test the stated hypothesis. Hence, in vitro correlates of EHV-1 myeloencephalopathy and neurologic disease shall be established. To test the hypothesis of transfer of pathogenicity between EHV-1 strains, we propose three specific aims:
Specific Aim 1: To induce a point mutation in an infectious bacterial artificial chromosome (BAC) clone of the non-neurologic ("abortion-only")EHV-1 strain NY03, and then restore the original, parental sequence in an independent mutant clone. The opposite set of point mutations has already been introduced based on the neuropathogenic strain Ab4 [18], which we propose to characterize in parallel with these new recombinant viruses.
Specific Aim 2: To characterize the generated mutant viruses with respect to their sensitivity to polymerase-targeting antiviral drugs in vitro. This will be measured by multi-step growth kinetics, with infectious viral titer compared with viral genome copies as measured by quantitative real-time PCR.
Specific Aim 3: To test if inducing the neuropathogenic allele increases replication rates in equine T lymphocytes, relative to B cells and monocytes directly ex vivo. Peripheral blood leukocytes from healthy horses will be cultured and infected with GFP-expressing ORF30 recombinant viruses. Cells will be sorted into leukocyte subpopulations by positive selection, and viral replication measured by quantitative real-time PCR.
The long-term goal of the project is to obtain a true understanding and appreciation of the contribution of 1) levels of viremia and 2) cellular tropism of EHV-1 strains to neuropathogenicity. Ultimately we believe that knowledge of these relationships will help us in generating efficacious preventive and therapeutic measures.
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