The goal of this project is to identify and characterize viral genes that control virulence and/or mediate immune evasion in the African clawed toad (Xenopus laevis) using frog virus 3 as a model. This project uses new technologies that selectively knock down the expression of specific viral genes, and generate mutant viruses in which specific candidate genes have been deleted. Mutant viruses will then be used to infect toads and the role of the candidate genes in disease and immune evasion determined. For example, it is likely that host has developed mechanisms to recognize and eliminate invading viruses, and that, in turn, viruses encode proteins designed to block those defenses. Such arms races are important in the evolution of both the host and the pathogen. If this is correct, then viral mutants where these countermeasures are deleted will fail to cause disease in infected animals. Successfully completed, this project will shed light on viral genes that control pathogenesis and, optimistically, reveal pathways used by the host to contain and eventually eliminate virus infections. In addition, this project may also uncover ways in which defects in amphibian immunity contribute to amphibian declines by making otherwise resistant animals susceptible to infection. Finally, this study may enhance understanding of the evolution of the immune system, and provide insight into ways to protect endangered amphibians from viruses and other pathogens. This project also has broader implications for the overall problem of worldwide decline in amphibian populations due in part to disease. It will provide training opportunities to a new generation of investigators in this field which is needed. Integral to the project is an outreach component to investigators at one of this nation?s Historically Black Colleges to enhance broadening participation in the field.
The goal of our recently completed NSF award (07-42711) was to determine the function of viral genes controlling the virulence of ranaviruses, a group of viruses that are increasingly infecting amphibians and contributing to their disappearance worldwide. Virulence genes are of two types: (1) those that interfere with the host’s immune response, and (2) those that, although not required for replication in most actively growing cells or tissues, enhance virus replication under conditions, or in certain cell types, that restrict viral growth. For this investigation, we used a model virus, frog virus 3 (FV3, family Iridoviridae) and a model host, Xenopus laevis. We chose FV3 because it is the best characterized member of a family of large DNA-containing viruses that target invertebrates and cold-blooded vertebrates. The African clawed frog, Xenopus laevis, was selected because it possesses the best characterized amphibian immune system at the molecular and cellular levels. We showed initially that viral gene expression can be selectively silenced using antisense morpholino oligonucleotides and siRNA. Specifically we demonstrated that targeted reductions of greater than 90% in the levels of the major capsid protein and the largest subunit of the viral homolog of RNA polymerase II impaired the formation of virions. Furthermore, by silencing the expression of these two viral genes we were able to determine the functions of their encoded proteins. To further ascertain the function of putative virulence genes during infection of an animal in vivo, we developed a methodology that used homologous recombination coupled with a powerful dual selection system based on drug resistance and fluorescence detection to selectively and stably knockout non-essential genes. Using this method, we successfully knocked out the viral genes that encode the 18K immediate early protein, the truncated FV3 homolog of eukaryotic translational initiation factor 2 alpha, as well as two other putative virulence genes (vCARD and beta-HSD) viral homologs, respectively, of a caspase activation and recruitment domain-containing protein and an hydroxysteroid dehydrogenase. As expected, deficiency in 18K and vIF-2 alpha had no impact on the ability of the recombinant viruses to replicate in cell culture indicating that they were "non-essential" genes. However, both knockout mutants displayed a ten-fold reduction in their ability to replicate in X. laevis tadpoles indicating that deficiency of either the 18K or vIF-2 alpha gene resulted in the production of attenuated virus. Based on these results, we are currently attempting to use this approach to knockout other putative virulence genes. Our intention is to ultimately identify novel viral genes that are critically involved in the incredible success of ranaviruses in infecting many different species of amphibians, fish and reptiles. This work will allow us to target virulence genes and optimistically generate attenuated viruses that can be used to protect endangered amphibian and fish species from iridovirus infections. This work will also shed light on the origins of the vertebrate immune system and on those specific aspects of the immune system of lower vertebrates that are critical for protection against viral disease.
