Micro-NMR and Nanoparticle Amplification                              for Botulinum Toxin                                              Diagnostics

Recombinant Envelope Protein Domain III as a                              Candidate Subunit Dengue                               Vaccine

A Highly Sensitive, Low-labor Pathogen Detector                              Based on Retroreflector-                              linked Immunosorbent                              Assay

Genetic Screens to Identify the Ebola Virus Receptor

High-throughput Assay Development Against                              Cryptosporidium Glycotlytic                              Enzymes

Model for Oral Ingestion of Ricin Toxin

Genetic Screens to Identify the Ebola Virus Receptor

Institution: Baylor College of Medicine, Houston, Texas

Principal Investigator: Richard E. Sutton, Ph.D.

Collaborators:
a) Mary K. Estes, Ph.D. – Baylor College of Medicine, Houston, TX
b) Jean Patterson, Ph.D. – Southwest Foundation for Biomedical Research, San Antonio, TX

Expected Product: Discovery of mechanism of Ebola virus binding and entry into cells, leading to the development of a vaccine and therapeutics.

Description: Ebola virus (EBO) is a member of the filoviridae and is the cause of fatal hemorrhagic fevers. It is currently categorized as both a BSL4 and class A bioterrorism agent. It is a filamentous, enveloped RNA virus, with a wide cellular tropism, both in terms of cell types and mammalian species. At present, no prophylactic vaccine yet exists for EBO and there are no effective therapies for infected patients. EBO encodes a precursor glycoprotein (GP) that is processed into GP1 and GP2. It is thought that GP1 mediates the initial binding of EBO to cell surfaces. Several years ago, folate receptor ??(FR?) was identified as a putative cofactor for EBO entry. Viral entry was inhibited by both soluble folate and folate binding protein, but recent published data suggests that FR? plays little if no role in EBO entry and it is likely there are other, as of yet uncharacterized, cellular factors that are involved in this process. This two-year WRCE developmental project seeks to characterize further EBO binding and entry into cells in two related but separate aims.
The first specific aim is to utilize a short hairpin (sh) RNA retroviral vector library introduced into EBO-susceptible cells to identify one or more cell clones specifically resistant to EBO infection. The corresponding cDNA clone(s) will be recovered and introduced into non-permissive T cells to determine whether it confers susceptibility to EBO, both as pseudotyped particles but also as replication-competent virus, the latter being performed under BSL4 conditions at Southwest Foundation for Biomedical Research (San Antonio) in collaboration with Jean Patterson. cDNA(s) will also be tested for their ability to mediate spread of Marburg (MAR) virus.
In the second aim (performed in parallel to the first) a genetic strategy using HIV-pseudotyped particles will be employed to identify genes that may mediate EBO binding and entry. A representational cDNA library will be introduced into an EBO non-permissive B cell line, and then the modified non-permissive cells subjected to infection with EBO GP pseudotyped viral particles. Candidate cDNAs will be further characterized and tested as described under Aim 1. It is hoped that these studies will allow a better understanding of filovirus binding and entry into cells, perhaps resulting in insights into filovirus pathogenesis in man and leads in small molecule inhibitors of this deadly viral pathogen.