'Transcriptional, taxonomic, and biochemical studies of two ranaviruses (Frog virus 3 and a Bohle iridovirus-like agent) infecting amphibians'.

摘要

Emerging infectious diseases that affect ectothermic species have become a major source of interest in the last decade due to their economic and ecological significance. Among emerging infectious diseases, iridovirus-associated mortality has been reported world-wide and appears to have a considerable impact on diverse species. Members of the family Iridoviridae are a diverse group of large dsDNA viruses that infect a wide range of invertebrate and cold-blooded vertebrate hosts. Frog Virus 3 (FV3, genus Ranavirus) is the type species and encodes approximately 100 proteins. Most of what is known about iridovirus replication has been learned from studies using FV3 as a model. Although FV3 is well characterized, the functions of only a third of the proteome are known and this is mostly due to sequence homology inference with other viral or cellular proteins. In this dissertation, three aspects of ranavirus biology are addressed: the host transcriptional response to FV3 infection, the identification of a Bohle iridovirus-like agent in North America, and four studies using biochemical, immunological and genetic approaches to ascertain viral protein function and intracellular location.;The first objective was to determine the transcriptional response of fathead minnow (Pimephales promelas) cells to infection with wild type FV3 and a knock out FV3 mutant lacking the 18 kDa immediate early gene. Transcriptional changes were monitored at 8 hr post-infection using a 60,000 feature FILM microarray. Marked upregulation of multiple immune-related gene transcripts including interferon (IFN) and IFN-stimulated genes (ISGs) was observed indicating that virus infection results in a robust anti-viral cellular response. This innovative tool can be used to provide a comprehensive view of how host cells respond to FV3 infection.;The second objective was to investigate the phylogeny of a novel ranavirus that was responsible for a recent disease outbreak in a North American amphibian collection. Whole genome sequencing was used to demonstrate that the causative agent was a virus closely related to Bohle iridovirus, a ranavirus previously detected only in Australia. Furthermore, sequence analysis showed the usefulness of using select ranavirus coding regions, e.g., a region encoding a neurofilament protein homologous to FV3 ORF32R, to differentiate among closely related virus isolates. The high mortality observed here demonstrated the severe impact such outbreaks have on survival assurance populations and suggests that survival assurance populations should be housed apart from other amphibians to minimize the risk of transmitted disease.;The last objective was ongoing attempts to identify new viral gene function and intracellular location using contemporary molecular or cellular approaches. Transmission electron microscopic analysis was used to examine the ability of temperature-sensitive mutants defective in viral DNA synthesis to form viral assembly sites at non-permissive temperatures. This study indicates that even in the absence of full viral DNA synthesis, viral assembly sites can form. In subsequent studies we used antisense morpholino oligonucleotides to knock down the expression of several putative viral genes and determined that all were essential for full virus replication. For example, knock down of a putative NTPase (ORF9L) resulted in a nearly 70% reduction in virus yield, whereas knock down of a protein of unknown function (ORF 41R) triggered a greater than 90% reduction in yield. In a related study, we generated mutants in which the genes for viral the homologs of the large subunit of ribonucleotide reductase (RRalpha) and LITAF (LPSinduced TNFa activating factor), were knocked out. These two genes were selected because RRalpha may play a role in increasing nucleotide pools and enhancing viral replication and LITAF may be involved mitochondria-mediated apoptosis. PCR analysis suggests that homologous recombination successfully replaced the targeted gene with the gene encoding enhanced green fluorescent protein (EGFP). Selection of fluorescent plaques will permit the isolation of KO mutants free of wild type virus and allow us to ascertain the role of each gene in replication. Lastly, a viral homolog of a caspaseactivation and recruitment domain (CARD) containing protein (vCARD) was cloned into a bacterial expression vector and the expressed protein was used to generate polyclonal rabbit anti-vCARD sera. Using immunohistochemistry, vCARD protein was detected predominantly within the cytoplasm of infected cells. This location would allow vCARD to interact with cellular detectors of viral RNA such as RIG-1 /MDA-5 (and/or MAVS/IPS-1) to short circuit the signaling pathway and block the induction of IFN.