Research Programs

 

 

 

 

 

 

 

 

 

 

 

 

 

Emerging infections: Principles of emergence

Program Leader: Mark Denison

 

SE-RP-001: Platforms for synthesis and testing of emerging zoonotic viruses

Mark Denison

Vanderbilt University
Ralph Baric

University of North Carolina

Bats represent approximately 20% of all mammalian species, and are increasingly recognized as vectors for virus evolution and transmission to other species, including humans. A large and constantly increasing number of Bat coronaviruses are being identified, including many with genetic similarity to known human coronaviruses of different groups, specifically SARS-CoV. The overall goals of this project are to dissect the molecular mechanisms of cross-species transmission of coronaviruses, and to develop platform strategies platform that will allow rapid response to natural, inadvertent, or intentional introduction of these viruses into the human population. The Aims remain unchanged from Year 1. Specific Progess in Year 2 resulted from a close collaboration of the Denison and Baric laboratories. A) Using information from sequence databases, we completed the design of four Bat-CoV genomes from viruses that are non-cultivatable and that represent different virus groups and genetic distance from SARS-CoV. The complete cDNA genome fragments for these viruses were commercially synthesized and are being assembled for transcription and recovery in culture in the coming year. B) We reported the synthesis and recovery of a previously noncultivatable human coronavirus (HKU1) in human airway epithelial cell (HAE) culture, establishing a platform for difficult-to-recover viruses. C) Spike genes that cover the majority of the range of identified mammalian coronaviruses were synthesized and will be used to test for virus entry in a Bat-CoV background and to develop broadly neutralizing polyclonal and monoclonal antibodies. D) The mutator phenotype of SARS-CoV nsp14 ExoN mutants was reported, demonstrating probable RNA proofreading and establishing a model for experimental evolution studies of coronavirus adaptation and trans-species movement. Ongoing work is demonstrating that the ExoN mutator phenotype of SARS-CoV is attenuating in mice. In addition, we have demonstrated that codon deoptimization can attenuate replication and pathogenesis. Over the next year we plan: 1) to recover the bat viruses and determine their fitness and adaptation, and determine the blocks to replication of non-cultivatable viruses. 2)  Use computer modeling and in silico design approaches to predict and test changes in spike that result in trans-species movement of coronaviruses. The designed spike proteins will be tested in the Bat-SRBD background. 3) From the host side, we have obtained ACE2 receptors from different Asian bat species and will test the expression of these receptors and their ability to support productive infection of bat CoVs. 4) Finally, we are testing effect ExoN mutations in coronavirus replicase gene on the recovery and adaptation of bat genomes, in order to define conserved interactome networks and broadly applicable attenuating strategies.

SE-NO-001: A genetic systems approach to host-pathogen interactions in orthopoxvirus infections

(Funding for this project ended March 1, 2010)

Richard Moyer

University of Florida

The orthopoxviruses, smallpox (variola), ectromelia and to a lesser extent monkeypox virus have narrow host ranges but for others (vaccinia and cowpox viruses) the host range is much more extensive. In light of bioterrorism concerns and the question of how viruses "jump" into new host species (both major concerns of SERCEB), it is imperative to study and identify host gene networks that determine and regulate host range. However, little is known about host genes that determine permissiveness, restriction or clinical course of disease. To elucidate such host genes, we will screen a panel of genetically diverse, C57BL/6J and DBA/2J advanced recombinant inbred (BXD ARI) mice for susceptibility to infection beginning with cowpox and ectromelia virus to establish the system and thereafter monkeypox virus. These mice and relevant services are provided by the SAID Core E. The relative permissiveness of a given virus, coupled with the genetic profiling of the infected versus the uninfected animals and an advanced bioinformatic analysis (genenetwork.org) will identify specific host genes/pathways which are critical for resistance/susceptibility of the infections and provide us a better understanding of the relationship between host and pathogen. These results will provide insight into the design of new intervention strategies, new targets for antiviral therapies and provide clues as to how "species jumping" might occur and identify combinations of biomarkers of host susceptibility/resistance genes that can be used for prognostic purposes and a platform for the screening of other pathogens. We propose the following specific aims: