Julie Metcalf

Bio: Julie Metcalf is an academic researcher from Trent University. The author has contributed to research in topics: Endosome & Aggresome. The author has an hindex of 5, co-authored 5 publications receiving 210 citations.

Comparative genomic analysis of the family Iridoviridae : re-annotating and defining the core set of iridovirus genes

Abstract: Members of the family Iridoviridae can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. In the present study, we describe the re-analysis of the Iridoviridae family of complex DNA viruses using a variety of comparative genomic tools to yield a greater consensus among the annotated sequences of its members. A series of genomic sequence comparisons were made among, and between the Ranavirus and Megalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, the Megalocytivirus genomes required the greatest number of altered annotations. Prior to our re-analysis, the Megalocytivirus species orange-spotted grouper iridovirus and rock bream iridovirus shared 99% sequence identity, but only 82 out of 118 potential ORFs were annotated; in contrast, we predict that these species share an identical complement of genes. These annotation changes allowed the redefinition of the group of core genes shared by all iridoviruses. Seven new core genes were identified, bringing the total number to 26. Our re-analysis of genomes within the Iridoviridae family provides a unifying framework to understand the biology of these viruses. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.

SIMPLE/LITAF expression induces the translocation of the ubiquitin ligase itch towards the lysosomal compartments.

Abstract: LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization.

Cellular LITAF Interacts with Frog Virus 3 75L Protein and Alters Its Subcellular Localization

Abstract: Iridoviruses are a family of large double-stranded DNA (dsDNA) viruses that are composed of 5 genera, including the Lymphocystivirus, Ranavirus, Megalocytivirus, Iridovirus, and Chloriridovirus genera. The frog virus 3 (FV3) 75L gene is a nonessential gene that is highly conserved throughout the members of the Ranavirus genus but is not found in other iridoviruses. FV3 75L shows high sequence similarity to a conserved domain found in the C terminus of LITAF, a small cellular protein with unknown function. Here we show that FV3 75L localizes to early endosomes, while LITAF localizes to late endosomes/lysosomes. Interestingly, when FV3 75L and LITAF are cotransfected into cells, LITAF can alter the subcellular localization of FV3 75L to late endosomes/lysosomes, where FV3 75L then colocalizes with LITAF. In addition, we demonstrated that virally produced 75L colocalizes with LITAF. We confirmed a physical interaction between LITAF and FV3 75L but found that this interaction was not mediated by two PPXY motifs in the N terminus of LITAF. Mutation of two PPXY motifs in LITAF did not affect the colocalization of LITAF and FV3 75L but did change the location of the two proteins from late endosomes/lysosomes to early endosomes.

Accumulation of endogenous LITAF in aggresomes

Abstract: LITAF is a 161 amino acid cellular protein which includes a proline rich N-terminus and a conserved C-terminal domain known as the simple-like domain. Mutations in LITAF have been identified in Charcot-Marie tooth disease, a disease characterized by protein aggregates. Cells transfected with cellular LITAF reveal that LITAF is localized to late endosomes/lysosomes. Here we investigated the intracellular localization of endogenous LITAF. We demonstrated that endogenous LITAF accumulates at a discrete cytoplasmic site in BGMK cells that we identify as the aggresome. To determine the domain within LITAF that is responsible for the localization of LITAF to aggresomes, we created a construct that contained the C-terminal simple-like domain of LITAF and found that this construct also localizes to aggresomes. These data suggest the simple-like domain is responsible for targeting endogenous LITAF to the aggresome.

Characterization of the promoter activity of a poxvirus conserved element.

Abstract: The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly con...

Ecology and pathology of amphibian ranaviruses.

Abstract: Mass mortality of amphibians has occurred globally since at least the early 1990s from viral pathogens that are members of the genus Ranavirus, family Iridoviridae. The pathogen infects multiple amphibian hosts, larval and adult cohorts, and may persist in herpetofaunal and oste- ichthyan reservoirs. Environmental persistence of ranavirus virions outside a host may be several weeks or longer in aquatic systems. Transmission occurs by indirect and direct routes, and includes exposure to contaminated water or soil, casual or direct contact with infected individuals, and inges- tion of infected tissue during predation, cannibalism, or necrophagy. Some gross lesions include swelling of the limbs or body, erythema, swollen friable livers, and hemorrhage. Susceptible amphi- bians usually die from chronic cell death in multiple organs, which can occur within a few days fol- lowing infection or may take several weeks. Amphibian species differ in their susceptibility to rana- viruses, which may be related to their co-evolutionary history with the pathogen. The occurrence of recent widespread amphibian population die-offs from ranaviruses may be an interaction of sup- pressed and naive host immunity, anthropogenic stressors, and novel strain introduction. This review summarizes the ecological research on amphibian ranaviruses, discusses possible drivers of emer- gence and conservation strategies, and presents ideas for future research directions. We also discuss common pathological signs of ranaviral disease, methods for diagnostic evaluation, and ranavirus surveillance methods. Inasmuch as ranaviral disease is listed as a notifiable disease by the World Organization for Animal Health and is a threat to amphibian survival, we recommend that biosecu- rity precautions are implemented by nations to reduce the likelihood of transporting ranavirus virions among populations. Biosecurity precautions include disinfecting footwear and equipment that comes in contact with surface water inhabited by amphibians and testing commercially shipped amphibians for the pathogen. We also encourage natural resource organizations to establish routine surveillance programs for ranaviruses in wild amphibian populations.

Family Iridoviridae: Poor Viral Relations No Longer

Abstract: Members of the family Iridoviridae infect a diverse array of invertebrate and cold-blooded vertebrate hosts and are currently viewed as emerging pathogens of fish and amphibians. Iridovirid replication is unique and involves both nuclear and cytoplasmic compartments, a circularly permuted, terminally redundant genome that, in the case of vertebrate iridoviruses, is also highly methylated, and the efficient shutoff of host macromolecular synthesis. Although initially neglected largely due to the perceived lack of health, environmental, and economic concerns, members of the genus Ranavirus, and the newly recognized genus Megalocytivirus, are rapidly attracting growing interest due to their involvement in amphibian population declines and their adverse impacts on aquaculture. Herein we describe the molecular and genetic basis of viral replication, pathogenesis, and immunity, and discuss viral ecology with reference to members from each of the invertebrate and vertebrate genera.

Distribution and Host Range of Ranaviruses

Abstract: Ranaviruses are globally distributed pathogens in amphibian, fish, and reptile communities that appear to be emerging. Cases of ranavirus infection or disease have been confirmed in at least 105 amphibian species (18 families), 41 fish species (22 families), and 29 reptile species (12 families). Ranaviruses have been documented on all continents except Antarctica, and are frequently associated with mass die-offs. Host susceptibility differs among species, with some species harboring subclinical infections and likely serving as reservoirs for the virus, and other highly susceptible species amplifying the virus. Currently, there are six recognized species of ranavirus, and all are not equally pathogenic among hosts. Frog virus 3 (FV3) is the type species of the genus Ranavirus, and appears to be the most globally distributed species infecting ectothermic taxonomic across three vertebrate classes. International commerce involving subclinically infected ectothermic vertebrates undoubtedly has contributed to the global distribution and emergence of ranaviruses. Herein, we describe the global distributed species infecting ectothermic vertebrates across three taxonomic classes.

Virus genomes and virus-host interactions in aquaculture animals

Abstract: Over the last 30 years, aquaculture has become the fastest growing form of agriculture production in the world, but its development has been hampered by a diverse range of pathogenic viruses. During the last decade, a large number of viruses from aquatic animals have been identified, and more than 100 viral genomes have been sequenced and genetically characterized. These advances are leading to better understanding about antiviral mechanisms and the types of interaction occurring between aquatic viruses and their hosts. Here, based on our research experience of more than 20 years, we review the wealth of genetic and genomic information from studies on a diverse range of aquatic viruses, including iridoviruses, herpesviruses, reoviruses, and rhabdoviruses, and outline some major advances in our understanding of virus-host interactions in animals used in aquaculture.

The DNA virus Invertebrate iridescent virus 6 is a target of the Drosophila RNAi machinery

Abstract: RNA viruses in insects are targets of an RNA interference (RNAi)-based antiviral immune response, in which viral replication intermediates or viral dsRNA genomes are processed by Dicer-2 (Dcr-2) into viral small interfering RNAs (vsiRNAs). Whether dsDNA virus infections are controlled by the RNAi pathway remains to be determined. Here, we analyzed the role of RNAi in DNA virus infection using Drosophila melanogaster infected with Invertebrate iridescent virus 6 (IIV-6) as a model. We show that Dcr-2 and Argonaute-2 mutant flies are more sensitive to virus infection, suggesting that vsiRNAs contribute to the control of DNA virus infection. Indeed, small RNA sequencing of IIV-6–infected WT and RNAi mutant flies identified abundant vsiRNAs that were produced in a Dcr-2–dependent manner. We observed a highly uneven distribution with strong clustering of vsiRNAs to small defined regions (hotspots) and modest coverage at other regions (coldspots). vsiRNAs mapped in similar proportions to both strands of the viral genome, suggesting that long dsRNA derived from convergent overlapping transcripts serves as a substrate for Dcr-2. In agreement, strand-specific RT-PCR and Northern blot analyses indicated that antisense transcripts are produced during infection. Moreover, we show that vsiRNAs are functional in silencing reporter constructs carrying fragments of the IIV-6 genome. Together, our data indicate that RNAi provides antiviral defense against dsDNA viruses in animals. Thus, RNAi is the predominant antiviral defense mechanism in insects that provides protection against all major classes of viruses.