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Since the successful use of vaccinia virus (VACV) in the immunization strategies to eliminate smallpox, research has been focused on the development of recombinant VACV strains expressing proteins from various pathogens. Attempts at decreasing the side effects associated with exposure to recombinant, wild-type viral strains have led to the development of attenuated viruses. Yet while these attenuated VACV’s have improved safety profiles compared to unmodified strains, their clinical use has been hindered due to efficacy issues in stimulating a host immune response. This deficiency has largely been attributed to decreased production of the target protein for immunization. Efforts to increase protein production from attenuated VACV strains has largely centered around modulation of viral factors, while manipulation of the translation of viral mRNAs has been largely unexplored. In this study we evaluate the use of translation enhancing element hTEE-658 to increase recombinant protein production in an attenuated VACV system. Optimization of the use of this motif is also attempted by combining it with strategies that have demonstrated effectiveness in previous research. We show that extension of the 5′ leader sequence containing hTEE-658 does not improve motif function, nor does the combination with other known translation enhancing elements. However, the sole use of hTEE-658 in an attenuated VACV system is shown to increase protein expression levels beyond those of a standard viral promoter when used with a wild-type virus. Taken together these results highlight the potential for hTEE-658 to improve the effectiveness of attenuated VACV vaccine candidates and give insights into the optimal sequence context for its use in vaccine design.

The iridovirus RNase III gene is one of 26 conserved core genes among the family Iridoviridae. Initial studies suggest this viral protein functions to suppress RNA interference pathways that may attack viral RNA during infection. Therefore, to determine if the Ambystoma tigrinum virus (ATV) RNase III-like gene (ORF 25R) can modulate the host innate immune response fish and human cells ectopically expressing 25R were treated with polyI:C and monitored for interferon synthesis and phosphorylation of eIF2α and PKR. We found a decrease in cellular IFN production and modulation of the PKR pathway. In addition, ATV deleted of the RNase III gene (ATVΔ25R) shows reduced pathogenicity in tiger salamanders. Collectively our data suggest that the ATV 25R protein is a pathogenesis factor that may function to help evade the host's immune response by masking activators of the IFN pathway.

Members of the genus Ranavirus (family Iridoviridae) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 – 155 kbp in length and they are predicted to encode around 90–160 genes. Currently, our knowledge of the function of ~50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV.

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

Ambystoma tigrinum virus (ATV) (family Iridoviridae, genus Ranavirus) was isolated from diseased tiger salamanders (Ambystoma tigrinum stebbinsi) from the San Rafael Valley in southern Arizona, USA in 1996. Genomic sequencing of ATV, as well as other members of the genus, identified an open reading frame that has homology to the eukaryotic translation initiation factor, eIF2α (ATV eIF2α homologue, vIF2αH). Therefore, we asked if the ATV vIF2αH could also inhibit PKR. To test this hypothesis, the ATV vIF2αH was cloned into vaccinia virus (VACV) in place of the well-characterized VACV PKR inhibitor, E3L. Recombinant VACV expressing ATV vIF2αH partially rescued deletion of the VACV E3L gene. Rescue coincided with rapid degradation of PKR in infected cells. These data suggest that the salamander virus, ATV, contains a novel gene that may counteract host defenses, and this gene product may be involved in the presentation of disease caused by this environmentally important pathogen.

The Iridoviridae is a family of large, icosahedral viruses with double-stranded DNA genomes ranging in size from 103 to 220 kbp. Members of the subfamily Alphairidovirinae infect ectothermic vertebrates (bony fish, amphibians and reptiles), whereas members of the subfamily Betairidovirinae mainly infect insects and crustaceans. Infections can be either covert or patent, and in vertebrates they can lead to high levels of mortality among commercially and ecologically important fish and amphibians. This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iridoviridae, which is available at www.ictv.global/report/iridoviridae.

Infectious diseases, including those caused by ranaviruses (family Iridoviridae), are among the suspected causes of global amphibian declines. Like many pathogens, ranaviruses appear to infect multiple species. We examined several North American amphibian ranavirus isolates to improve our understanding of the effects these viruses have on host communities. Our study had two objectives. The first was to characterize isolates from epizootics affecting wild amphibian populations and compare them to previously described isolates. The second was to test whether amphibian ranaviruses infect ecologically relevant heterologous species, and if so, document the outcome of exposure. The combined results of restriction endonuclease (RE) digestion analyses, sequence analyses, and experimental challenge trials suggest that two amphibian ranaviruses, Ambystoma tigrinum virus (ATV)-like viruses and Frog Virus 3 (FV3)-like viruses, are distinct viral species with different ecologies. Characterizations revealed th...

Distinguishing whether pathogens are novel or endemic is critical for controlling emerging infectious diseases, an increasing threat to wildlife and human health. To test the endemic vs. novel pathogen hypothesis, we present a unique analysis of intraspecific host-pathogen phylogenetic concordance of tiger salamanders and an emerging Ranavirus throughout Western North America. There is significant non-concordance of host and virus gene trees, suggesting pathogen novelty. However, non-concordance has likely resulted from virus introductions by human movement of infected salamanders. When human-associated viral introductions are excluded, host and virus gene trees are identical, strongly supporting coevolution and endemism. A laboratory experiment showed an introduced virus strain is significantly more virulent than endemic strains, likely due to artificial selection for high virulence. Thus, our analysis of intraspecific phylogenetic concordance revealed that human introduction of viruses is the mechanism underlying tree non-concordance and possibly disease emergence via artificial selection.

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.

Our understanding of origins and spread of emerging infectious diseases has increased dramatically because of recent applications of phylogenetic theory. Iridoviruses are emerging pathogens that cause global amphibian epizootics, including tiger salamander (Ambystoma tigrinum) die-offs throughout western North America. To explain phylogeographical relationships and potential causes for emergence of western North American salamander iridovirus strains, we sequenced major capsid protein and DNA methyltransferase genes, as well as two noncoding regions from 18 geographically widespread isolates. Phylogenetic analyses of sequence data from the capsid protein gene showed shallow genetic divergence (< 1%) among salamander iridovirus strains and monophyly relative to available fish, reptile, and other amphibian iridovirus strains from the genus Ranavirus, suggesting a single introduction and radiation. Analysis of capsid protein sequences also provided support for a closer relationship of tiger salamander virus strains to those isolated from sport fish (e.g. rainbow trout) than other amphibian isolates. Despite monophyly based on capsid protein sequences, there was low genetic divergence among all strains (< 1.1%) based on a supergene analysis of the capsid protein and the two noncoding regions. These analyses also showed polyphyly of strains from Arizona and Colorado, suggesting recent spread. Nested clade analyses indicated both range expansion and long-distance colonization in clades containing virus strains isolated from bait salamanders and the Indiana University axolotl (Ambystoma mexicanum) colony. Human enhancement of viral movement is a mechanism consistent with these results. These findings suggest North American salamander ranaviruses cause emerging disease, as evidenced by apparent recent spread over a broad geographical area.

Disease is among the suspected causes of amphibian population declines, and an iridovirus and a chytrid fungus are the primary pathogens associated with amphibian mortalities. Ambystoma tigrinum virus (ATV) and a closely related strain, Regina ranavirus (RRV), are implicated in salamander die-offs in Arizona and Canada, respectively. We report the complete sequence of the ATV genome and partial sequence of the RRV genome. Sequence analysis of the ATV/RRV genomes showed marked similarity to other ranaviruses, including tiger frog virus (TFV) and frog virus 3 (FV3), the type virus of the genus Ranavirus (family Iridoviridae), as well as more distant relationships to lymphocystis disease virus, Chilo iridescent virus, and infectious spleen and kidney necrosis virus. Putative open reading frames (ORFs) in the ATV sequence identified 24 genes that appear to control virus replication and block antiviral responses. In addition, >50 other putative genes, homologous to ORFs in other iridoviral genomes but of unknown function, were also identified. Sequence comparison performed by dot plot analysis between ATV and itself revealed a conserved 14-bp palindromic repeat within most intragenic regions. Dot plot analysis of ATV vs RRV sequences identified several polymorphisms between the two isolates. Finally, a comparison of ATV and TFV genomic sequences identified genomic rearrangements consistent with the high recombination frequency of iridoviruses. Given the adverse effects that ranavirus infections have on amphibian and fish populations, ATV/RRV sequence information will allow the design of better diagnostic probes for identifying ranavirus infections and extend our understanding of molecular events in ranavirus-infected cells.

The molecular size limitations of the digestive system, including the filter chamber of immature and adult Bemisia tabaci (Gennadius) B biotype (=B. argentifolii), were studied by tracking the movement of fluorescent-labeled molecules and microspheres ingested by whiteflies. Soluble fluorescent molecules and labeled dextrans, ranging from 389 to 2,000,000 Da, were observed throughout the digestive tract of immatures 10–30 min after feeding was initiated. After removal of labeled molecules from the diet, fluorochromes were cleared from digestive system of immatures within 2 h. Fluorescent-labeled 0.1- and 0.2-μm microspheres were ingested by larvae and saturated the digestive system within 2 h after initiation of feeding. Large, 0.5-μm spheres were not observed in the digestive tract of immatures, probably because singly or as aggregates, they were too large to enter the stylet food canal. The smallest spheres examined, 0.02 μm, were not detectable in the digestive tract of immatures. Observations...

The spatially compartmentalized replication strategy of ranaviruses and other members of the family Iridoviridae is unique among DNA viruses and is a hallmark of the family. Viral DNA replication initially takes place within the nucleus and results in the formation of genome-sized molecules. Subsequently, viral DNA is transported into the cytoplasm wherein a second round of DNA synthesis takes place in which concatemers 10–20-times the size of the viral genome are generated. As with viral DNA synthesis, viral RNA synthesis shows a similar compartmentalization: early transcription takes place within the nucleus in reactions catalyzed by host RNA polymerase II, whereas late viral transcription occurs within the cytoplasm in reactions catalyzed by a virus-encoded transcriptase. Morphologically distinct viral assembly sites form within the cytoplasm and viral DNA is packaged into virus particles by a headful mechanism resulting in genomes that are circularly permuted and terminally redundant. Although the outlines of viral metabolism and virion assembly are known, the functions of specific viral gene products remain to be elucidated. Recent attempts at understanding the precise role of viral proteins in these and other events within virus-infected cells are ongoing and have utilized a variety of contemporary approaches including gene knock down mediated by antisense morpholino oligonucleotides and RNA interference, knock out and conditionally lethal mutants, and ectopic expression of recombinant viral proteins.

Ranaviruses (genus Ranavirus, family Iridoviridae) are large double-stranded DNA (dsDNA) viruses that infect economically and ecologically important cold-blooded vertebrates worldwide. Taxonomically, ranaviruses belong to a monophyletic group of viruses referred to as the Nucleocytoplasmic Large DNA Viruses (NCLDV). The NCLDV cluster contains viruses with the largest known viral genomes and infects a diverse array of eukaryotic hosts. The family Iridoviridae is currently divided into five genera: Iridovirus, Chloriridovirus, Megalocytivirus, Lymphocystivirus, and Ranavirus. Ranavirus taxonomy is based on restriction endonuclease fragment length polymorphism (RFLP) profiles of genomic DNA, virus protein profiles, DNA sequence analysis, and host specificity as well as whole genome dot plot analysis and phylogenetic analysis of individual and concatenated gene sequences. In this chapter, we discuss the current taxonomy of the genus Ranavirus as well as the phylogenetic relationship among currently identified ranaviruses. In addition, we discuss the future of ranavirus taxonomy in light of current phylogenetic analyses.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.

Ambystoma tigrinum virus (ATV) is a lethal virus originally isolated from Sonora tiger salamanders Ambystoma tigrinum stebbinsi in the San Rafael Valley in southern Arizona. USA. ATV is implicated in several salamander epizootics. We attempted to transmit ATV experimentally to fish and amphibians by injection, water bath exposure, or feeding to test whether ATV can cause clinical signs of infection or be recovered from exposed individuals that do not show clinical signs. Cell culture and polymerase chain reaction of the viral major capsid protein gene were used for viral detection. Salamanders and newts became infected with ATV and the virus was recovered from these animals, but virus could not be recovered from any of the frogs or fish tested. These results suggest that ATV may only infect urodeles and that fish and frogs may not be susceptible to ATV infection.

A feeding chamber and diet were developed for rearing Bemisia argcntifolii (Bellows & Perring) from eggs to 4th in star under aseptic conditions. The feeding apparatus consisted of a plastic diet chamber covered with stretched Parafilm that was held in place by another piece of plastic and binder clips. Surface-sterilized eggs were placed on the Parafilm, and the chamber was incubated in a petri dish at 90-95% RH, 25-27oC and a photoperiod of 16:8 (L:D) h. The most successful diet consisted of 5% yeast extract in 30% sucrose. Developmental times of 1st-, 2nd-, and 3rd-instar nymphs were 4.6, 3.4, and 4.2 d, respectively, which is comparable to development times on plants. Although whitefly nymphs can be reared on this diet to 4th in star, complete development to the adult stage was not achieved. The utility of the feeding system was demonstrated in bioassay with Ivermectin.

The Sonora tlger salamander Ambystoma tignnuni stebb~nsi Lowe is a genetically d~stinct race restricted to about 30 small ponds in the San Rafael Valley 111 southern Arizona, USA, which was added recently to the USA Federal List of Endangered Species Populations of these salamanders periodically expenence decimating epizootics. Virus was ~solated from diseased salamanders using fish cell cultures, injected into healthy laboratory-reared salamanders, and then reisolated in cell culture. Electron microscopy of thin sections from dying salamanders revealed abundant enveloped and nonenveloped icosahedral virus particles approximately 160 to 180 nm in diameter in the cytoplasm of skin and liver cells and free in the intercellular spaces. This virus, believed to be an iridovirus based on viral morphology and host pathology, was demonstrated to be the primary pathogen in these epizootics, and is the first lethal epizootic virus reported from salamanders. We have named the virus Ambystoma tigrinum Virus (ATV). Hemolytic bacteria were isolated from sick individuals, but we were unable to induce the disease by exposing salamanders to isolated bacteria at concentrations up to 10' ml-'

Ranaviruses (family Iridoviridae , genus Ranavirus ) are large, double-stranded DNA (dsDNA) viruses whose replication is restricted to ectothermic vertebrates. Many highly pathogenic members of the genus Ranavirus encode a homologue of the eukaryotic translation initiation factor 2α (eIF2α). Data in a heterologous vaccinia virus system suggest that the Ambystoma tigrinum virus (ATV) eIF2α homologue (vIF2αH; open reading frame [ORF] 57R) is involved in evading the host innate immune response by degrading the interferon-inducible, dsRNA-activated protein kinase, PKR. To test this hypothesis directly, the ATV vIF2αH gene (ORF 57R) was deleted by homologous recombination, and a selectable marker was inserted in its place. The ATVΔ57R virus has a small plaque phenotype and is 8-fold more sensitive to interferon than wild-type ATV (wtATV). Infection of fish cells with the ATVΔ57R virus leads to eIF2α phosphorylation, in contrast to infection with wtATV, which actively inhibits eIF2α phosphorylation. The inability of ATVΔ57R to prevent phosphorylation of eIF2α correlates with degradation of fish PKZ, an interferon-inducible enzyme that is closely related to mammalian PKR. In addition, salamanders infected with ATVΔ57R displayed an increased time to death compared to that of wtATV-infected salamanders. Therefore, in a biologically relevant system, the ATV vIF2αH gene acts as an innate immune evasion factor, thereby enhancing virus pathogenesis.

Members of the genus Ranavirus (family Iridoviridae ) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.

Interferons were the first of the anti-viral innate immune modulators to be characterized, initially characterized solely as anti-viral proteins [reviewed in Le Page, C., Genin, P., Baines, M.G., Hiscott, J., 2000. Inteferon activation and innate immunity. Rev. Immunogenet. 2, 374-386]. As we have progressed in our understanding of the interferons they have taken a more central role in our understanding of innate immunity and its interplay with the adaptive immune response. One of the key players in function of interferon is the interferon-inducible enzyme, protein kinase (PKR, activatable by RNA). The key role played by PKR in the innate response to virus infection is emphasized by the large number of viruses, DNA viruses as well as RNA viruses, whose hosts range from insects to humans, that code for PKR inhibitors. In this review we will first describe activation of PKR and then describe the myriad of ways that viruses inhibit function of PKR.

What's your diagnosis?: Dermal Lesions, Hemorrhage, and Limb Swelling in Laboratory Axolotls